Acute Abuse of Tramadol Induces Intensive Pulmonary Histopathological Trauma in Rats - An Experimental Comprehensive Overview

Middle East Journal of Applied Science & Technology (MEJAST)
Vol.3, Iss.3, Pages 81-119, July-September 2020
ISSN: 2582-0974 [81] www.mejast.com
Country: Egypt
Acute Abuse of Tramadol Induces Intensive Pulmonary Histopathological
Trauma in Rats - An Experimental Comprehensive Overview
G. El-Sherif * & H. K. Abdel-Zaher
Department of Zoology, Faculty of Science, Minia University, Egypt.
Article Received: 23 June 2020 Article Accepted: 21 August 2020 Article Published: 22 September 2020
1. Introduction
Tramadol is known as the most popular analgesic around the world. Tramadol are now readily available to the
public so, fatal and dangerous symptoms appeared after tramadol abuse. These symptoms varied among loss of
consciousness, cardiac arrest, prolonged hospitalization due to tramadol metabolites poisoning, cardiac arrest and
death after subsequent complications.
As deaths after tramadol poisoning were generally recorded, mortality rate in young males was most prevalent.
However, more care for the elderly, in terms of risk of aspiration, which indicates a lack of defense mechanisms of
prevention of these complications. Therefore, prevention of intractable use of tramadol and care after poisoning
with tramadol is obvious. After the death, the highest diagnostic aid will be biological samples with drug poisoning,
first stomach contents and then urine samples, (Bita et al., 2015).
Moreover, Bita et al., (2015) discussed out of 49 cases dead, 80% men and 20% were women as significant
correlation was seen between recent use of tramadol and gender (P value <0.003). Cause of death has been
cardiopulmonary arrest (67%), convulsion (49%), decreased level of consciousness (47%) and acute respiratory
failure (12%).
But in a study by Matthiesen et al., (2010), they noticed that the most common cause of death was neurological
symptoms and there was significant correlation between the studied lung microscopic findings and the acute
poisoning of tramadol (P value</004%). Meanwhile, the most pathologic findings were seen less than 34 years old,
100% of patients who has pneumonia and aspiration signs were over 40 years.
The recent use of tramadol and gender significant correlation was found as (P <0.003). Most of deaths were
recorded in cases that used tramadol as acute abuse mode to suicide and died following its complications. In
ABSTRACT
Tramadol is a narcotic-like pain reliever used in medicine to treat moderate to severe pain in adults after an operation or a serious injury. It is also used
to treat long-standing pain when weaker painkillers no longer work. Tramadol works by blocking pain signals from travelling along the nerves to the
brain. It can still be addictive if abused, especially when taken for a long period of time (chronic) or when taken in larger doses (acute) as a narcotic
rather than a pain-killer. In this comprehensive histopathological study, the commercial tramadol hydrochloride prescribed in Egypt was used in both
forms, as a solution of 100 mg ampoules or as 225 mg tablets. Male adult Wistar rats (Rattus norvegicus) were used as experimental models that were
administered tramadol orally and injection for regular durations and calculated dose concentrations. Some reliable histological, histochemical,
immunocytochemical and ultrastructural techniques were applied to investigate the experimental manifestations and life-threatening signs/symptoms
of tramadol poisoning and to check the traumatic histopathological and biochemical impacts of tramadol abuse in acute administration modality on
lungs of rats. Many histopathological lesions e.g., toxicity Lymphocyte infiltration, hemorrhage, necrotic areas, cytoplasmic and membranous
degenerations, depletion or increase of some biomolecules, ultrastructure damages and immunocytochemical signs were recorded. These results were
described, evaluated and confirmed to a variety of recent studies. It was concluded that, use of tramadol as a pain-killer in medicine or as an abused
narcotic among addicts, should be controlled and we are in need for more extensive attention to the clinical and narcotic abuse of tramadol.
Keywords: Tramadol Acute dose, Rats Lungs, Respiration, Toxicity, Histochemical, Immunocytochemical, Ultrastructure.

reviewing tramadol toxicological impacts, blood, urine, tissues, gallbladder and stomach contents were clinically
investigated, there was a significant correlation between the recent use of tramadol and positive toxicity, all had
p-value <0.05. Most deaths caused by tramadol were 25-34 years old in males. Any of them didn’t have the history
of tramadol abuse or other disease in the past, and significant relationship between age and tramadol was seen (P
<0.05). So, tramadol administration complications and deaths reported in men were most common this age range.
Adverse reactions of therapeutic use of tramadol include nausea and dizziness (> 10%),drowsiness, fatigue,
headache, increased sweating, vomiting, dry mouth, constipation (1-10%), diarrhea, and cardiovascular
deregulations (palpitations, tachycardia, postural hypotension - particularly after rapid intravenous administration)
(0.1-1%). Respiratory depression, epileptiform convulsions, tremor, bradycardia, hallucinations, and anxiety are
rare (0.01-0.1%) (Jarernsiripornkul et al., 2003)
Withdrawal reactions include restlessness, agitation, anxiety, sweating, insomnia, hyperkinesia, tremor,
paresthesias and gastrointestinal symptoms, similar to opioid withdrawal symptoms (Ojha & Bhatia, 2010). The
incidence of adverse effects depends on the dose and the mode of administration (Barkin, 2008). Analysis of
French pharmaco-vigilance data from the period 1987-2006 indicated that the incidence of adverse reactions to the
tramadol-paracetamol combination was 44.5 cases per 105 patient-years, which was significantly higher than for
the dextro-propoxyphene-paracetamol combination (24.9) and the codeine-paracetamol combination (12.5)
(Tavassoli et al., 2009).
The potential for respiratory depression is commonly associated with opioid analgesics and occurs due to a
decrease in the sensitivity of the respiratory center to carbon dioxide. This results in a decreased tidal volume and
respiratory rate (Lee et al., 1993).
Opiates reduce the sensitivity of the respiratory center to carbon dioxide. This may result in decreased tidal volume
and decreased respiratory rate. Because of the μ-opioid agonist activity of O-desmethyl-tramadol, tramadol may
lower the respiratory rate and potentially lead to severe respiratory depression. This has incidentally been observed
in overdose cases of tramadol (Wang, 2009). However, at therapeutic doses tramadol is not likely to cause
significant respiratory depression (Grond & Sablotzki, 2004).
In healthy subjects, tramadol reduced the sensitivity to carbon dioxide, but did not reduce the ventilator response to
hypoxia (Warren, et al., 2000). At therapeutic doses, tramadol produced less respiratory depression, both in adults
and in children, compared to morphine, pethidine, and oxycodone (Tarkkila, et al., 1998). Regarding overdose
situations, tramadol may produce life-threatening respiratory depression (Shipton, 2000).
In Egypt, it was found that 20% of Egyptian male students have used drugs and among them 25% have continued to
do so (Soueif et al., 1986). Among secondary school male students, 5.05% abused hashish, 0.84% abused opiates,
2.72% abused tranquilizers, 1.79% abused stimulants, and 2.26% abused hypnotics (Soueif et al., 1990). The last
National Survey report stated that 9.6% of Egyptians used drugs at least once during their lives (Hamdi et al.,
2013). Tramadol abuse has dramatically increased in Egypt since 2008 and has led to many admissions to addiction
treatment centers (Abo-elmaged et al., 2013). In a recent study, about 40% of temporary cleaners and 21% of
permanent cleaners working in governmental hospitals in Zagazig, Egypt, used tramadol (Abbas et al., 2013).

Experimentally, there are many published studies regarding effects of different doses of tramadol on rat's lungs.
These investigations were based on checking the painkiller characteristics of tramadol, its role in maintenance of
cellular regeneration in rat lungs, prevention of lung tissue injury after skeletal muscle ischemia-reperfusion and
tramadol ability to cause alterations in cell morphology, inflammatory cell infiltrates and cell death in brain, lung
and heart of Wistar rats. These studies were found to study the analgesic properties of tramadol rather than its abuse
effects as a narcotic drug among drug-addicts all over the world.
It was found that tramadol prevented the effect of surgery on natural killer cell activity and metastatic colonization
in lungs of rats (Gaspari, et al., 2002) compared to morphine as analgesics. However, Samaka et al., (2012),
concluded that stem cell markers are the main modulators of life saving as they re-expressed early in response to
cell injury by toxicity and late in maintenance of cellular regeneration in rat lungs, by playing crucial roles
throughout the journey. They also stated that activation of Nestin and Notch 1 signaling in both acute and chronic
tramadol toxicity groups might provide a molecular basis for potential protective and treatment strategies.
On the other hand, tramadol was found to prevent lung tissue injury after skeletal muscle ischemia-reperfusion
(Takhtfooladi, et al., 2013). Moreover, in histological analyses, tramadol and tapentadol were found to cause
alterations in cell morphology, inflammatory cell infiltrates and cell death in brain, lung and heart of Wistar rats,
although tapentadol caused more damage than tramadol (Faria et al., 2017).
2. Aim of the Work
In this study, we are aiming to evaluate all possible experimental manifestations and life-threatening
signs/symptoms of tramadol poisoning. In addition, we tried to check the traumatic histopathological and
biochemical impacts of tramadol abuse in acute administration modality on lungs of rats. This was to notify the
severity of side effects of the acute therapeutic doses of tramadol and its abuse effects as a narcotic drug among
drug-addicts in Egypt as well.
3. Materials & Methods
3.1 Drugs
a. Tramadol (Tramadol Hydrochloride, 100mg ampoules) was obtained as a solution from ADWIA Co.S.A.E; 10th
of Ramadan City - Egypt.
b. Tramadol Hydrochloride Bp-225mg was used as tablet.
c. All other chemicals were of analytical grade and were purchased from standard commercial suppliers.
3.2 Experimental Animals
A total of 120 male adult rats (Rattus norvegicus) weighing approximately 180-200g were used in the present
study (Animal house of the Faculty of Agriculture Suppliers, Minia University, Minia, Egypt).
3.3 Experimental Protocols
Modes of administration

Daily oral administration mode of tramadol tabs suspended in saline was applied, by using Intra-gastric gavage. It
was also administered as intra-peritoneal injection of tramadol solution daily for a month, rats were sacrificed after
10, 20, 30 days respectively (Table 1).
Table 1: Acute Mode of Tramadol Administration
Intra-peritoneal Injection Oral Administration via Intra-gastric Gavage
S.No.
Group
No.
of
rats
Dose
Tramadol HCL 100 mg/2 ml
Ampoules in Saline Solution
Duration
No.
of
rats
Dose
Tramadol HCL 100 mg/ Tablet
Suspended in Saline Solution
Duration
1 Group I a 5 20 mg (T HCL 1 ml/day) 10 days 5 20 mg (T HCL 1 ml/day) 10 days
2 Group I b 5 40 mg (T HCL 1 ml/day) 10 days 5 40 mg (T HCL 1 ml/day) 10 days
3 Group I c 5 80 mg (T HCL 1 ml/day) 10 days 5 80 mg (T HCL 1 ml/day) 10 days
4 Group I d 5 Control (1 ml/day Saline Solution) 10 days 5 Control (1 ml/day Saline Solution) 10 days
5 Group II a 5 20 mg (T HCL 1 ml/day) 20 days 5 20 mg (T HCL 1 ml/day) 20 days
6 Group II b 5 40 mg (T HCL 1 ml/day) 20 days 5 40 mg (T HCL 1 ml/day) 20 days
7 Group II c 5 80 mg (T HCL 1 ml/day) 20 days 5 80 mg (T HCL 1 ml/day) 20 days
8 Group II d 5 Control (1 ml/day Saline Solution) 20 days 5 Control (1 ml/day Saline Solution) 20 days
9 Group III a 5 20 mg (T HCL 1 ml/day) 30 days 5 20 mg (T HCL 1 ml/day) 30 days
10 Group III b 5 40 mg (T HCL 1 ml/day) 30 days 5 40 mg (T HCL 1 ml/day) 30 days
11 Group III c 5 80 mg (T HCL 1 ml/day) 30 days 5 80 mg (T HCL 1 ml/day) 30 days
12 Group III d 5 Control (1 ml/day Saline Solution) 30 days 5 Control (1 ml/day Saline Solution) 30 days
Acute experimental study
Animal were divided randomly into seven groups, 30 animals in group 1 and 15 rats in each group 2-7, and treated
as follows:
Group 1: Comprised 30 rats, served as control groups. There were divided into six groups. Three groups each
consists of 5 rats administered oral dose of saline solution for ten, twenty, and thirty days by using gavage. Others
15 rats, also were divided into three groups and injected intra-peritoneal with saline for ten, twenty, and thirty days.
Rats sacrificed after ten, twenty, and thirty days.
Group 2: Comprised 15 rats, five rats were daily administered oral dose of tramadol Hcl suspended in saline
solution equal to 20mg/Kgb.wt./day for ten days, other five rats was daily administered oral dose 40mg/kgb.wt./day
for ten days, other five rats was daily administered oral dose 80mg/kgb.wt./day for ten days . This group sacrificed
after ten days.

Group 3: Comprised 15 rats, five rats were daily administered oral dose of tramadol Hcl suspended in saline
solution equal to 20mg/Kgb.wt./day for twenty days, other five rats was daily administered oral dose
40mg/kgb.wt./day for twenty days, other five rats was daily administered oral dose 80mg/kgb.wt./day for twenty
days. This group sacrificed after twenty days.
Group 4: comprised 15 rats, five rat were daily administered oral dose of tramadol Hcl suspended in saline
solution equal to 20mg/kgb.wt.day for thirty days, other five rats was daily administered oral dose
40mg/kgb.wt./day for thirty days, other five rats was daily administered oral dose 80mg/kgb.wt./day for thirty days.
This group sacrificed after thirty days.
Group 5: comprised 15 rats, five rats were daily injected intra peritoneally with tramadol Hcl ampoule equal to
20mg/Kgb.wt./day for ten days, other five rats was daily injected dose 40mg/kgb.wt./day for ten days, other five
rats was daily injected dose 80mg/kgb.wt./day for ten days. This group sacrificed after ten days.
Group 6: comprised 15 rats, each 5 rats was daily injected intra peritoneal with tramadol Hcl ampoule equal to
20mg/Kgb.wt./day for twenty days, other five rats was daily injected dose 40mg/kgb.wt./day for twenty days,other
five rats was daily injected dose 80mg/kgb.wt./day for twenty days. This group sacrificed after twenty days.
Group 7: comprised 15 rats, each rat was daily injected intra peritoneal with tramadol Hcl ampoule equal to
20mg/Kgb.wt./day for thirty days, other five rats was daily injected dose 40mg/kgb.wt./day for thirty days ,other
five rats was daily injected dose 80mg/kgb.wt./day for thirty days. This group sacrificed after thirty days.
3.4 Histological & Histochemical Studies
A- Heamatoxylin and Eosin
The fixative used for the routine heamatoxylin and eosin stain was 10% buffered formalin (Lillie, 1954).
B- Carbohydrates demonstration
General carbohydrates were demonstrated following of (Hotchkiss, 1948) Periodic Acid-schiff (PAS).
C- Total proteins demonstration
Total proteins were visualized by using the Mercury Bromphenol Blue method (Mazia et al., 1953).
D- Connective tissue demonstration
Connective tissue were investigated by using Mallory Triple Stain method (Krichesky,2009)..
3.5 Ultra-structural Studies
A-Primary fixation
For transmission electron microscopy, small pieces of lungs were fixed in 2.5% glutaraldehyde buffered with .1M
cacodylate (pH7.2) at room temperature for 2 hours. Fixation continued at 4°C for slows down autolysis processes
and reduce tissue shrinkage.
B-Post fixation

Specimens were washed three times with the same buffer and post fixed in phosphate-buffered 1% osmium
tetroxide for 2 hours at room temperature then dehydrated in ascending grades of ethanol.
3.6 Immuno-histochemical Studies
Sections were taken on positive slides and immunostained using avidin-biotin technique Zarnescu and Brehar
2008.
4. Results
1- Morphological changes
Rat that were treated with 40mg/kg of tramadol injected for 20 days showed sever hemorrhage patches in left and
right lungs (Fig. 1). Lungs appeared enlarged with irregular and convoluted surfaces in left lobe with hemorrhage
and irregularly contracted surface in right lobe increased (Fig. 2). Increasing dose of tramadol and increasing time
of administration caused relatively more hemorrhage in lungs such as in group IIc, when rats were treated with
80mg/kg. of tramadol orally for 20 days (Fig. 3). After one month, hemorrhage covered all lungs surfaces as in
group IIIb at which rats were treated with 40mg/kg. of tramadol orally and injection ( Figs. 4 & 5). Increasing dose
up to 80 mg/kg through oral administration, lungs appeared more congested and proliferated as seen in (Fig. 6).
However, in the same group - when rats injected with 80 mg/kg tramadol – Lungs revealed scattered focal nodule
(Fig. 7) filled with pus appeared as green mucus substance (Fig. 8).
2- Histological & Histopathologal finding
A. Control group
The epithelial cells at the ends of all the twigs form the "respiratory units". The pleural cavity is lined by simple
squamous mesothelium. The conducting passageways of the respiratory bronchi and bronchioles are lined
by pseudo stratified epithelium, which is ciliated and includes mucus-secreting goblet cells. Incoming particulates
(dust, bacteria) adhere to the mucus, which is then swept upward and away by the cilia (Figs. 9 & 10). Because the
passage of air depends on always open passageways, bronchi are supported by skeletal elements in the form of
rings made of cartilage. An extensive vascular plexus allows heat-exchange to adapt air temperature before it
reaches the delicate alveoli. As in other mucous cells, the nucleus is compact and intensely stained (basophilic)
with H&E (Fig. 9 & 10).
B. Treated groups
Increased infiltration of lymphocytes appeared if the dose increased as in group Ib, when rats were treated with
40mg/kg of tramadol injected for 10 days. Haemorrhage also appeared scattered between lungs septa and rounded
bronchi (Fig.11), noticed also increased infiltration of lymphocytes. Haemorrhage was noticed also in (Fig.12) at
which it was characterized by accumulations of extravasated blood cells in airways, around pulmonary vessels, and
in alveolar lamina with neoplasia, necrosis, and inflammation.
Keratinizing cysts, or the "squamous cysts" and "epidermal cysts" also exhibited squamous metaplasia of the
alveolar epithelium, cystic keratinizing epithelioma (Figs.13 & 14). Alveolar epithelial hyperplasia appeared in

(Fig.15) in rats which were treated with 40mg/kg of tramadol orally for 20 days, with alveolar macrophages that
may be associated with primary hyperplasia.
Acute inflammation, (Fig.16), with predominant infiltrating lymphocytes, fewer macrophages and neutrophils were
also recorded. There were also evidences of oedema or hyperaemia. Lymphocytes were also predominating
in chronic active inflammation (Figs.17 & 18), but there are also a significant number of neutrophils. Both lesions
may contain macrophages.
Grossly, these lesions would be characterized by the presence of supportive inflammation with pus (Fig. 19). The
tissue surrounding the exudate contains fibroblasts, fibrous connective tissue, and mixed inflammatory cells,
depending on the chronicity of the lesion. Lymphocytes appeared predominant in chronic inflammation (Fig. 20 &
21).
In group IIIb, rats were treated with 40mg/kg of tramadol orally for 30 days showing thickened septa filled with
congested capillaries, infiltration of lymphocytes surrounding an alveolus, macrophage, fibrosis and intestinal
hemorrhage (Fig. 22).
Some regenerating cells e.g., cytomegaly, karyomegaly, cytoplasmic basophilia, increased nuclear to cytoplasmic
ratio of Atypia cells appeared in group IIIc, when rats were treated with 80mg/kg of tramadol orally for 30 days
(Fig. 23 & 24) .
A small, focal accumulations of inflammatory cells - without evidences of inflammation e.g., edema, hemorrhage,
cell swelling, degeneration, or necrosis, alveolar septal thickening or fibrin deposition - were diagnosed as "cellular
infiltration cellular" rather than inflammation (Figs.25 & 26).
3- Histochemical test for total proteins
A. Control group
The protein contents of the lungs cells of control rats (Fig. 27) were demonstrated by the mercuric bromophenol
blue method as blue granules against a light-blue ground cytoplasm, which indicate the presence of some soluble
proteins. The protein granules were scattered all over the cytoplasm. The cells were limited by intensely stained
plasma membranes. The nuclear envelopes and nucleoli as well as some chromatin elements were also positively
stained with heavy stained interstitial septa.
B. Treated groups
An increased total protein contents appeared more pronounced on the 20 and 30 days following the treatment of an
increased tramadol dose. Animals received tramadol dose of 80mg/kg orally for 30 days revealed lower protein
contents in lungs cells. Cells and its nuclear membranes were also stained higher than the normal condition (Fig.
28).
Lungs of rats injected with tramadol 40mg/kg. for 30 days demonstrated marked increase of protein contents in
comparison to the lungs of the control groups (Fig. 29).The cytoplasm exhibited highly bromphenol blue reaction
with some scattered fine and moderately sized granules lying mainly near the cell and nuclear membranes. Highly

increased protein contents in lungs cells appear in group IIIc rats that were treated with 80mg/kg of tramadol
injection for 30 days (Fig. 30).
4- Histochemical examination for polysaccharides
A. Control group
Lungs of control rats stained with PAS showed normal content of glycogen particles that appeared as deeply red
purple colour PAS positive inclusions densely located inside the cytoplasm. Most of the PAS positive products
were displaced laterally towards one side of the cell (glycogen migration/flight phenomenon) caused by the effects
of the fixative applied. All the nuclei of lungs cells acquired positive stain ability which indicates the existence of
PAS-positive materials (Fig. 31).
B. Treated groups
Lungs of rats treated with 40mg/kg of tramadol orally for 30 days showed less increased stained with PAS positive
materials if compared to the control animals (Fig. 32).
However, rats treated with tramadol as 40 mg/kg injected dose for 30 days showed a marked increase in glycogen
content of the pulmonary cells especially in those located in the peripheral lobular areas (Fig. 33).
In addition, rats injected with 80 mg/kg for 30 days showed also highly increase in glycogen content of the lungs
cells especially in those located in the peripheral lobular areas. The cytoplasm exhibited highly PAS positive
reaction with some scattered fine and moderately sized granules lying mainly near the cell and its nuclear
membranes. Progressive increased of glycogen contents in lungs cells appear in group IIIc at which rats were
treated with 80mg/kg of tramadol orally and injected for 30 days (Fig. 34).
5- Connective tissue fibres
A. Control group
Examination of lungs of control rats stained with M.T.S (Mallory Triple Stain) showed normal content of
connective tissue fibres, which appeared as finely blue-coloured fibres located outside the pulmonary cells. Most of
the M.T.S. positive products were displaced laterally towards outside of the cell caused by the effects of the fixative
on the tissue. None of the nuclei of all lungs cells acquired any positive stain (Fig. 35).
B. Treated groups
Faintly increase in connective tissue fibres in lungs cells stained with M.T.S. was noticed in group IIc (rats were
treated with 80mg/kg of tramadol orally for 20 days (Fig. 36). While marked increase of connective tissue fibres
replaced lungs cells and stained with. M.T.S appeared in-group IIIb, rats that were treated with 40mg/kg of
tramadol orally for 30 days (Fig. 37). Highly increased of connective tissue fibres replaced lungs cells appeared in
groups IIIb, when rats injected with 40 mg/kg tramadol for 30 days (Fig. 38). Progressive increase of connective
tissue fibers replaced lungs cells and air sacs was founded in group IIIc, at which rats were treated with 80mg/kg of
tramadol orally and injected for 30 days. Lungs appeared collapsed and tight (Fig. 39).

6- Ultrastructural findings
A. Control group
Lung consists of an alveolar region (parenchyma) and non-parenchyma conducting airways (which are part of the
anatomic dead space) and larger vessels. The airways branch in irregular dichotomy into the lung together with the
arteries, thus defining broncho-arterial units “inside-out” (from the hilum to the periphery). In the most distal
branching generations, alveoli are connected to the airways.
Clusters of alveoli are arranged in functional units termed acini. An acinus is a blind-ending parenchymal unit
beginning with a transitional (i.e. the first generation of an alveolated) bronchiole. Within an acinus, all airways
(alveolar ducts and respiratory bronchioles) have alveoli attached to their walls and. Actually, the “wall” of alveolar
ducts consists of a network of alveolar openings. It separates the air compartment (alveolar airspace) from the blood
compartment (capillary lumen). (Fig. 40).
B.Treated groups
In group Ic, that treated with 80mg/kg of tramadol orally for 10 days, many RBCs appeared engulfed by
macrophages. Autophagosomes and Autolysosomes appeared in macrophages and connective tissue fibrosis
appeared as well in bundles near macrophage. Hypertrophic smooth endoplasmic reticulum (SER) in type II
lunge-cells and endothelial cells appeared swollen. Extensions of the swollen cells are sheltering the red blood cell
(RBCs). Natural killer cells with large granules also found (Fig. 41).
While in-group Ib, (rats were treated with 40mg/kg of tramadol injection for 10 days) activated macrophages with
lysosomes appeared. Plasma cells with autolysosomes were illustrated as large nucleated cells. Some cytoplasmic
electron-dense deposits and several multi vesicular bodies also found with disorganized phospholipid
membranes and amorphous content inserted. Others macrophage appeared with segmented nucleus.
Collagenous fibrous and elastic fibers increased (Fig.42).
In addition, when rats were treated with 80mg/kg of tramadol injection for 10 days, activated macrophage
appeared. Mitochondria in macrophage lungs cells were swollen with disrupted or disintegrated cristae and the
osmiophilic lamellar bodies were fusion or disappeared. Glycogen accumulation was visualized in the alveolar
space. Some cytoplasmic electron-dense deposits and several multi vesicular bodies with disorganized
phospholipid membranes and amorphous content was inserted. Necrotic cells appeared as completely
degeneration of nucleus. Apoptotic cells also found with irregular shape nucleus (Fig. 43).
Basophilic cells with large granular cytoplasm were found in group IIc as rats were treated with 80mg/kg of
tramadol orally for 20 days. Macrophage with hypertrophic smooth endoplasmic reticulum and degeneration of
rough endoplasmic reticulum also appeared. Apoptotic cells with degenerated nucleus were also found in (Fig.
44). Cytoplasm was found vaculated with electron-dense deposits and several multi-vesicular bodies with
disorganized cellular membranes with amorphous contents.
Increased infiltration of red blood cells appeared in group IIc, at which rats were treated with 80mg/kg of
tramadol injection for 20 days. Apoptotic lungs cells type I nuclei appeared with irregular shape nucleus with

ruptured cell membrane and mostly vacuolated cytoplasm. Elastic fibers appeared surrounded apoptotic nuclei
(Fig. 45).
Large macrophage with autophagosomes & autolysosomes appeared in group IIIa, (rats were treated with 20mg/kg
of tramadol orally for 30 days). Others macrophage cells with pseudopodia appeared with irregular shape nucleus
surrounded with fibroblast. Apoptotic lungs cells type I appeared with proliferation rough endoplasmic
reticulum with irregular shape nucleus and necrosis (expressed as tail arrows). Increased necrosis combined
with increased cytoplasmic vacuolation was found (Fig. 46).
Infiltration of natural killer cells with dark granular cytoplasm and irregular shape nucleus appeared in animals
of group IIIa, (rats were treated with 20mg/kg of tramadol orally for 30 day). Necrotic lungs cells with nuclear
disappear and residual cells contents were illustrated in the same group. Apoptotic cells with picnotic nuclei &
proliferation of smooth endoplasmic reticulum appeared together with marked fibrosis surrounding the necrotic
cells (Fig. 47).
Activated macrophages with autophagosomes & autolysosomes appeared in group IIIb, (rats were treated with
40mg/kg of tramadol orally for 30 days). Glycogen accumulation can be visualized in the alveolar space with few
multi vesicular bodies inserted. Hypertrophic rough endoplasmic reticulum, some cytoplasmic electron-dense
deposits and several multi vesicular bodies with disorganized phospholipid membranes and amorphous content
were noticed. Apoptotic cells appeared with irregular shape nuclear membrane. Mitochondria were found
swollen with disintegrated cristae and the osmiophilic lamellar bodies were fused or disappeared causing
mitochondrial damage. Collagenous and elastic fibers increased and surrounded the alveoli (Fig. 48). Atabia cells
appeared in lungs in group IIIc, rats were treated with 80mg/kg of tramadol orally for 30 days characterized by
cytoplasmic basophilic, hyperchromatic nuclei, loss of nuclear polarity, high nuclear to cytoplasmic ratio,
karyomegaly, anisokaryosis, and pleomorphism. Macrophage appeared engulf erythrocytes and irregular shape
nucleus. Necrotic type II lungs cells with degeneration of cells content also appeared in (Fig. 49). Lungs septa
appeared extended and swollen with irregular shape and proliferated endothelial cells. The nucleus of lungs cells
type II appeared proliferated and segmented. Vacuolated cytoplasm filled with foreign substance was found
increased. Hyper trough of rough endoplasmic reticulum appeared in endothelial cells. Mitochondria of lung cells
were found swollen with disrupted or disintegrated cristae and osmiophilic lamellar bodies that were fused or
disappeared. Collagen fibers were accumulated in the lungs septa (Fig. 50). Injected rats for 30 days with 80mg/kg
of tramadol illustrate increased the engulfed red blood cells with macrophage. Red blood cells appeared flatten and
irregular in shape. Lungs septa were found swollen and surrounded with collagenous fibrosis (Fig. 51).
7- Immunohistochemical observations
A. Control group
Immunohistochemical (IHC) method was applied to visualize the distribution and amount of molecules in lung
tissues using specific antigen-antibody reaction. Lungs section of control animals revealed normal lungs cells
alvuoli with no detectable immunostaining for caspase-3 (Fig. 52).

B. Treated groups
Lungs section from all treated groups showed moderate to severe intense caspase-3 immunostaining. In group IIc &
IIIc, (rats were treated with 80mg/kg of tramadol orally for 20 &30 days respectively) a moderate Caspase
3-positive reaction in lungs cells appeared (Fig 53 & 54).
Also moderate Caspase 3-positive reaction scattered in lungs cells showed in group IIc, rats were treated with
80mg/kg of tramadol injected for 20 days (Fig. 55).
While intensive Caspase 3-positive reaction scattered in lungs cells recorded in group IIIc, rats were treated with
80mg/kg of tramadol injected for 30 days (Fig. 56).
5. Discussion
1-Histopathological light microscopic findings
Through this comprehensive study, it was found that trmadol causes emphysema (abnormal enlargement of lung
espesially the alveoli with loss of pulmonary elasticity) which is characterised with irreversible airspace
enlargement, followed by a decline in lung function. The imbalance between elastase and anti-elastase activity,
rupture of alveolar walls, and inflammation in the lung parenchyma are some of the hallmarks of this disease
(Rocha, N.d., 2017). In addition to the well-known impact of emphysema on lungs, extrapulmonary effects have
also been described, such as pulmonary arterial hypertension, and changes in right ventricular structure and
function; skeletal muscle wasting; and body weight loss.
These systemic manifestations are associated with increased risk of exacerbation and decreased survival which is in
an agreement with Agusti et al., (2003). The current therapeutic approach for emphysema is mainly focused on the
use of tramadol drugs (Vogelmeier et al., 2017). To date, there has been no effective therapy able to modify the
long-term decline in lung function. Therefore, a new pharmacological therapy able to reduce inflammation and
remodelling, as well as mitigate the extrapulmonary effects associated with emphysema, might represent a
potential disease-modifying strategy.
Administration of tramadol that induces emphysema experimentally is more advantageous than cigarette smoke
exposure, as tramadol is inexpensive and able to induce greater and more widespread lung damage (Suki et al.,
2003). Both models may result in cachexia and pulmonary hypertension , but the lung damage induced by tramadol
for longer after induction in contrast to cigarette smoke (Barreiro et al., 2012). In addition to lung structural damage
induced by tramadol, a persistent lung inflammatory process with electrolysis and fibro genesis has been observed.
The specific toxic substance-induced emphysema has been shown to produce loss of lean and total body mass,
likely suggesting cachexia (Oliveira et al., 2016).
Lung inflammation is one of the hallmarks of emphysema. Contributing to this feature, alveolar macrophages can
be activated by several extracellular signals to polarize into the M1 or M2 phenotypes. At the early stages of
inflammation, macrophages are functionally distinct from those at later stages. Early-phase macrophages are
predominantly M1-biased cells and contribute to extracellular matrix deposition and fibrosis, likely producing

pro-fibrotic cytokines. During the late resolution phase, macrophages tend to be alternatively activated,
remodelling-competent, M2-biased macrophages (Lee et al., 2012).
Acute lung injury (ALI) with tramadol is a highly lethal inflammatory pulmonary disorder. During the onset of
ALI, many types of damaging factors promote apoptosis of pulmonary vascular endothelial cells and alveolar
epithelial cells, increasing lung tissue damage and contributing to the ALI inflammatory response. Apoptosis plays
an important pathogenic role (Martin et al., 2003). ALI and acute respiratory distress syndrome (ARDS) can be of
either pulmonary (direct) or extra pulmonary (indirect) origin (Perl et al., 2011). Based on available evidences,
direct and indirect ALI are truly different processes (Kovach & Standiford, 2012). While there is substantial
overlap, many studies estimated that approximately 55% of ARDS is caused by direct, rather than indirect, lung
injury (Calfee et al., 2007).
The present study indicates that the major morphologic alteration at the alveolar capillary level in hemodynamic
pulmonary edema is interstitial in location and focal in nature. The apparent discrepancy between the lung weight
ratios and the light microscopic appearance of the lungs is because the edema fluid in this preparation has a low
protein content and hence is not stained by hemotoxylin or eosin. The expanded collagen-containing regions of the
septum seem to represent sites of fluid accumulation in the tissues even though transudates per se cannot be
visualized by current electron microscopic techniques. This focal change is in direct contrast to the diffuse changes
observed after tratment with tramadol toxic substances (Finecold, 1967).
With these toxic agents the edema fluid is not limited to the collagen-containing regions of the interstitial spaces but
is found in all portions of the septum and is associated with degenerative changes in the endothelium and
epithelium. In terms of Starling's hypothesis for the exchange of fluid across capillary walls, the formation of
edema in the present experiments involving increased capillary hydrostatic pressure and decreased colloid osmotic
pressure may be regarded merely as an accentuation of the normal process of fluid exchange in the lungs rather than
a pathologic one (Greene, 1965).
It is then conceivable that the collagen-containing interstitial areas of the alveolar septum could serve as a reservoir
to collect excess fluid which enters the interstitial space. These connective tissue areas, in addition to providing the
structural framework of the lung, could then also serve to bind edema fluid in a state of relatively low potential
energy and operate as a "sponge" for maintaining the alveolus in a dry state (Knudsen & Ochs 2018). As a corollary
to this idea, free fluid in an alveolus or accumulation of fluid throughout the interstitial space would imply prior
saturation of the local fluid-binding sites.
Also, the observations of Uhley et al., (1961) on the delay before lymph flow from the lung increased in
hemodynamic pulmonary edema which is consistent with the sequence of water binding to collagen, overflow into
interstitial spaces and gradual accumulation of fluid in the perivascular spaces and lymphatic system. It seems clear
that diffusion across the airblood barrier is not severely compromised by pulmonary edema, and that arterial
unsaturation follows accumulation of fluid in small bronchioles and alveoli rather than from diffusion limitation
(Said et al., 1964).

The present study has shown that the thin and thick portions of the alveolar capillaries differ markedly with respect
to the accumulation of edema fluid. The possibility then arises that the good oxygenation reflects the effective
operation of the thin part of the capillary in gas exchange even though diffusion through the opposite wall may be
somewhat impeded by thickening.The focal collections of fluid observed in the present study of hemodynamic
edema provide no new information concerning the pathway of fluid movement from capillary lumen to collecting
sites which is in need for more investigatios.
The vascular markers used were invariably found within capillaries and never in association with the edema fluid.
The fact that both the endothelial cells and their intercellular junctions retained their normal structure in
hemodynamic edema does not preclude fluid movement across these cells or their junctions (Luft, 1965). In fact, it
is even conceivable that the interstitial fluid may not come from the capillaries. For example, as has been suggested
by Staub et al.,(1967), it may originate from larger vessels as perivascular edema which then extends down to the
alveolar level. However, this possibility remains to be examined by electron microscopy, since the resolution
available with light microscopy may be inadequate to demonstrate the earliest collections of edema fluid within
alveolar septa. Also, the presence of numerous red blood cells within the interstitium of the lung and the alveolar
spaces in hemodynamic pulmonary edema, even though vascular markers do not seem to escape, has still to be
explained.
In any case, the pink color of the edema fluid in these animals is due to the escape of erythrocytes into the alveolar
spaces, while the white edema fluid of the alloxan-treated animals reflects the retention of the erythrocytes within
the vascular spaces.The origin and significance of the cytoplasmic fragments within the interstitium is unclear
although they may represent fragments of endothelial or smooth muscle cells (Cottrell et. al., 1967).
Although the present observations have provided some insight into the morphological aspects of hemodynamic
pulmonary edema, they have also emphasized that much remains to be learned concerning the routes of exchange
for fluids and particulate matter between the blood vessels,the interstitium, the alveolar spaces and the lymphatics
of the lungs.
In rats, the sloughing of the alveolar epithelium is associated with alveolar pulmonary oedema, capillary
congestion and a mild acute inflammatory reaction appeared. Eosinophilic hyaline membranes are also common.
Alveolar pulmonary oedema during the early stage of tramadol poisoning in animals is often sufficiently extensive
to cause severe dyspnoea. Alveolar pulmonary oedema, capillary congestion, hyaline membranes and an acute
inflammatory exudate are also found.It is commonly accepted that the granular pneumocytes secrete pulmonary
surfactant and, therefore, destruction of these cells by toxican would lead to loss of surfactant with a corresponding
increase in surface tension of the alveolar fluid. This could then withdraw fluid from the alveolar capillaries to
produce oedema. Such an increase in surface tension has been demonstrated in animals (Robertson et al , 1971).
These authors also suggest that loss of pulmonary surfactant leads to the formation of hyaline membranes, and for
this reason they propose tramadol poisoning as an experimental model for the idiopathic respiratory distress
syndrome. In the experimental model of tramadol-induced emphysema used in this study, an increased
inflammation was established, which increased the macrophage counts, increased collagen fibre content in the

alveolar septa and pulmonary vessel walls, increased elastic fibre content in the alveolar septa, incresed alveolar
hyperinflation and collapse, and disfunction of lung mechanics. Damage in the capillaries is often seen with
sub-endothelial and endothelial oedema, followed by necrosis of endothelial cells, which results in nude basement
membrane. More serious injuries often involve the presence of high numbers of inflammatory cells in the septa and
damage to the epithelium (primarily epithelium type I), which results in alveolar oedema (often along with hyaline
membrane formation). These were confirmed to the extensive and prolonged injury or severe epithelial damage is
also often associated with stimulation of fibroblasts and collagen deposition as previously described (Adamson,
1990).
It is possible that epithelium damage occurs in isolated areas and/or that the oedema is at least partly due to reduced
activity of epithelium sodium channels (Hee et al., 2011). In this study, rat lungs showed damage to the
endothelium, namely endothelial swelling with distended cytoplasmic extensions and thickening of the endothelial
basement membrane. The presence of oedema, blood and debris in the alveolar spaces was observed. Red blood
cells in the alveolar space suggests breakdown of barriers and that epithelial damage is occurring.
Capillary congestion was one of the most prominent observations by both light microscopy and electron
microscopy. It is likely that this congestion results in inefficient blood flow and blood gas exchange in the lungs.
In addition to RBC: endothelium binding, another factor that may contribute to capillary congestion is the
endothelium swelling, especially with the endothelium extensions cutting through the capillary lumen. It is
hypothesized that the swelling and extensions are an indication of activated endothelium and/or microthrombi
organization.The presence of various leukocytes in the lung’s capillaries, septa and alveolar spaces suggests that
inflammation plays an important part in the pathogenesis of lung injury in this model.
Monocyte, macrophages and neutrophils have been identified previously by electron microscopy in lung injury.
Monocytes and macrophages are consistently observed in studies and often contain haemozoin indicating
phagocytosis of infected cellsTaylor et al.,(2012), and their presence could result in the production of
pro-inflammatory products that could also contribute to endothelial injury (Chua et al., 2013).
The observation of an alveolar macrophage with vesicles filled with crystals (likely haemozoin) suggests that
alveolar macrophages play a role in clearing deposite from the alveolar space. Neutrophils have been highlighted as
playing a role in severe injury caused by tramadol toxic substance, possibly by contributing to pulmonary lesions
(Senaldi et al., 1994).Lung fibrosis occurs in some lung injury (Carr et al., 1999) with toxic substance in lungs
injury especially in high doses of tramadol.These may be due to damaged of epithelial cells followed by cerrosis
and fibrosis.
Over a period of 20 to 30 days after treatment with tramadol, infiltration of inflammatory cells, dominated by
neutrophils and macrophages, was observed in lung tissue. Histological sections of the lungs taken from rats treated
with 40 mg/kg and 80 mg/kg of tramadol formed acute alveolitis consisting of an increased number of
particle-containing neutrophils and macrophages, intra-alveolar hemorrhage, exudation of serum protein into the
alveolar spaces, and diffuse deposition of particles at the bronchi and in alveolar epithelial cells.

The intensity of the inflammatory changes in the lung and the number of neutrophils in the treated rats appeared to
increase with both time after exposure and increased doses. Acute and chronic pulmonary toxicity induced by
tramadol is characterized by purulent inflammation, thickening of alveolar walls, fibrosis, and formation of
granulomas. In the normal lung tissues, type 1 alveolar epithelial cells and vascular endothelial cells are closely
packed together across the basement membrane (Naota et al., 2010).
In this study, the destruction of the alveolar walls in the inflammatory lesions was observed by electron
microscopy. The degeneration and detachment of type 1 alveolar epithelial cells, and the dissociation of the
basement membrane between type 1 alveolar cells and endothelial cells were observed in the pulmonary lesions of
the mice treated with tramadol (Kaewamatawong et al., 2005).
Gil & Martinez-Hernandez (1984) added that laminin, which is present in the pulmonary basement membranes and
is used as a marker for normal alveolar structures, plays a central role in the stability of basement membranes, as
well as in the control of cellular interactions. In this study, inflammatory lesions in the lung tissues showed a
decrease in laminin immunopositivity resulting from the destruction of the pulmonary tissues, including the
basement membrane. Alpha-SMA is a marker for smooth muscle fibers, as well as myofibroblasts. Myofibroblasts
have a role in the repair of injured tissue by producing connective tissue components during the chronic phase of
inflammation (Darby et al., 2016). The increase in α-SMA immunopositivity observed in the lung inflammatory
lesions in the mice treated with tramadol. TNF-α plays important roles in acute inflammation, such as activation of
inflammatory cells and induction of secondary tissue injury.
The expression of chemokines is modulated by the presence of TNF-α (Pryhuber et al., 2003). TNF-α is produced
by activated macrophages, lymphocytes, and endothelial cells, as well as other various cell types (Wajant et al.,
2003). IL-6, which has multiple functions, is produced by many cell types, such as T lymphocytes, monocytes,
endothelial cells, and fibroblasts. The release of inflammatory cytokines, such as TNF-α and IL-6, in pulmonary
lesions was observed in animals cell types, such as T lymphocytes, monocytes, endothelial cells, and fibroblasts
(Tanaka & Kishimoto, 2014). The release of inflammatory cytokines, such as TNF-_ and IL-6, in pulmonary
lesions was observed in animals treated with tramdol.
NO and reactive oxygen species are reported to cause severe oxidative stress (Porter et al., 2006).The activation of
iNOS in the inflammatory cells and the production of NO were reported to be associated with silica-induced
damage of the lung (Zeidler et al., 2004). Tramadol is a potent respiratory irritant, while the mechanism by which
tramadol metabolized formed its cytotoxic effects. Tramadol reacts directly with tissue constituents, and
cytotoxicity is presumably a function of this reactivity (Kimbell et al., 2001). The pulmonary changes pronounced
in this work as hemorrhages, thickened alveolar wall, dilatation of the pulmonary blood vessels and inflammatory
cells invasion were consistent with the findings in the rats lungs after exposure to20& 40&80mg/kg of tramadol
(Neelam et al., 2011).
The mechanism of polymorphonuclear leukocytes inflammatory cells invasion induced by tramadol explained by
Ryoko et al.,(2010), who reported that tramadol rapidly increased vascular permeability in rat airway and produced
microvascular leakage in the airway through stimulation of tachykinin NK1 receptors by tachykinins released from

sensory nerves. There was emphysema evident from bulla formation of air spaces in all animal exposed groups due
to rupture of inter alveolar septa. This finding obtained in the experiment of (Njoya et al., 2009) who reported that
the mechanism by which tramadol brought about the ulceration of the alveoli was by excavation and desquamation
of the surtramadolce epithelium and derangement with distorted supporting tissues of alveolar wall.
The ulceration was observed to be dependent on the days of exposure as the animals exposed for longer days
presented a more sever ulceration. Massive cellular proliferation of bronchiolar epithelium were detected, the
epithelial lining of bronchioles showed loss of mucosal folds and the cellular proliferation resulted in conversion of
the epithelial lining of bronchi from pseudostratified columnar ciliated epithelium into thickened hyperplastic
bronchiolar epithelium formed of many layers of cells. Also, Monticello et al. (1996) whom observed an increase in
cell proliferation in the respiratory tract and hyperplastic epithelial changes following repeated exposure to
tramadol.
Appearance of multiple mitotic figures, cell vesicularity in bronchiolar epithelium indicated carcinogenicity. Naya
& Nakanishi, (2005) reported that tramadol is carcinogenic at the site of contact as a consequence of epithelial cell
regenerative proliferation resulting from cytotoxicity and mutation. In the present study invasion of pulmonary
tissues with spindle shaped fibroblast cells forming granulamatous pneumonic areas was observed. The mechanism
of fibrosis induced by tramadol explained by Valérie et al., (2012) who reported that interleukin-11 (IL-11) is a
molecular target of tramadol which could be involved in inflammatory and fibrogenic pulmonary effects in a
dose-dependent manner in cultured lung epithelial cells.
Squamous metaplasia had developed in more than one layer of bronchiolar epithelium, squamous metaplasia was
identified as hyperplastic epithelium formed of large polygonal cells. These data are in agreement with Ohtsuka et
al., (1998) who had observed that after rats taken tramadol, for 10 days changes such as degeneration, necrosis
stratification and squamous metaplasia were observed in bronchi of the lungs.
Tramadol exposure resulted in the appearance of Atypia cells which could not be found in the pulmonary
epithelium of the nonexposed rats. These cells were observed in many pathological conditions, in the bronchioles
of nitrogen dioxide exposed rats (Anderson et al., 1977) in tracheobronchial epithelium of hamster receiving
intratracheal instillations of benzopyrene (Harris et al., 1974), in bronchi of hyper oxygenated guinea pig (Torikata
et al., 1976), in nasal mucosa of workersemployed in a nickel refinery (Boysen & Reith, 1980) and in
tracheobronchial epithelium of colchicine treated rabbit (Ohashi et al., 1991). Although the Atabia cells was
described by many authors, the genesis of these tunnels could not be determined until now. However, different
authors described different explanations of Atabia cells origin. Tandler et al., (1983) had demonstrated the
intracytoplasmic vacuoles to be extremely long tunnels that were connected to the epithelial surface.
However, Tandler & Liedtke (1981) considered the Atabia to be formed by invagination of the epithelial surface.
This consideration is confirmed by the results of the present study, the intracellular Atabia could be considered as a
huge dilation of rough endoplasmic reticulum as the wall of Atabia cell was unilaminar and resembled the
membranes of the adjacent rough endoplasmic reticulum. Moreover, the small clear dilated vesicles of the rough
endoplasmic reticulum appeared to coalesce with the large Atabia cells. The results obtained in this study,

pertaining to the relationship between histological changes and exposure duration, are in agreement with those
studies obtained by (Javedan et al., 1999) on rat nasal mucosa, and Davarian et al., (2005) who determined the
histological changes of albino rat tracheal mucosa exposed to tramadol.
Pulmonary neutrophilia is a characteristic of several inflammatory lung conditions. Experimental pulmonary
exposure of tramadol injected and oral administration induces a fast and intense neutrophil response (Domenici et
al., 2004). Under normal physiological conditions, neutrophils are silently and swiftly eliminated through
apoptosis, followed by phagocytosis by alveolar macrophages (Cox et al., 1995). However, if the phagocytosis
system fails, apoptotic cells die through secondary necrosis, a pro-inflammatory event associated with cell
membrane disruption and extracellular spreading of cell contents such as mucous (Haslett, 1999).
Moreover, Medan et al. (2002) suggested that secondary necrosis takes place in inflamed lungs, these authors
found the peak of apoptotic cells in bronchoalveolar lavage (BAL) to be followed by an increase in activity of the
pan-necrosis marker lactate dehydrogenase (LDH).While this important finding suggests secondary necrosis to be
a significant feature of tramadol induced lung inflammation. Special focus was given to neutrophil infiltration and
clearance, due to the possibility of finding direct evidence for secondary necrosis of neutrophils to be a significant
feature of the inflammation.
This approach allowed the current authors to follow the fate of cells within patchy areas of intense neutrophil-rich
inflammation and infiltration (hereafter referred to as inflammatory foci (IF)) at various phases of lung
inflammation in detail, and to study the clearance processes following cell death. The data revealed that the
extensive number of cells within IF were neutrophils, of which large numbers were undergoing apoptosis and
secondary necrosis, suggesting neutrophils to be the primary source of LDH during an intense lung inflammation.
Taking the pathogenic potential of necrosis into consideration (Liu et al., 2003), this study suggests neutrophil
secondary necrosis to be a potential pathogenic mechanism during an intense neutrophil-rich lung inflammation.
It is clear, however, that secondary necrosis occurs extensively in the airway lumen of rat’s lungs with chronic
obstructive pulmonary disease, as assessed by TEM analysis of directly fixated sputum samples (Erjefalt et al.,
2004). Hence, secondary necrosis seems to be a common fate of senescent and apoptotic cells trapped in airway
mucus plugs.
To what extent such luminal necrosis affects the underlying airway mucosa is currently unknown, although it has
recently been demonstrated in vitro that neutrophils undergoing secondary necrosis have the capacity to damage
airway epithelial cells Liu et al., (2003), suggesting a potentially pathogenic role of luminal secondary necrosis.
From the present study, it can be concluded that the most likely site for secondary necrosis to occur is in areas of
intense inflammation and neutrophil infiltration (inflammatory foci). These results agreed with Rydell-Tomanen et
al., (2006) which revealed that the vast majority of the macrophages within (inflammatory foci) contain multiple
large phagosomes and had occasionally started to disintegrate in necrosis.
Macrophages are the most common inflammatory cells found in the lung after injury. The close proximity of
fibroblasts and macrophages may be important in limitation of fibrosis after lung injury (Sibille & Reynolds, 1990).
It has been demonstrated that stimulation of alveolar macrophages by tramadol may enhance the process of lung

fibrosis induced by bleomycin (Chyczewska et al., 1993). Macrophages, especially in an active form, can release
many cytokines (Kelley, 1990). The tumour necrosis factor-alpha (TNF-a), interleukine-I (IL- I), fibroblast growth
factor (FGFs) and platelet-derived growth factor (PDGF) are known to be stimulators of cell proliferation. TNF-a
and IL-I, acting separately, stimulate the increase in fibroblasts in the lungs, while their joint action inhibits cell
divisions (Kelley, 1990).
It has also been demonstrated that local macrophage proliferation is an important process in interstitial lung
diseases (Pforte et al., 1993). Our observations in TEM also suggest the possibility of AM contribution to the
stimulation of fibroplasia processes. This refers mainly to the experimental group IIIc, where the greatest collagen
cumulation was observed, especially in the region of rebuilt lung parenchyma as well as the cumulation of alveolar
macrophages, frequently demonstrating morphological traits of increased activity. Also, ultrastructural pictures of
alveolar macrophages in group IIIb confirm a stimulating effect of toxic substance upon these cells.
A stimulatory effect of macrophages on fibroplasia processes in the course of experimental lung emphysema seems
possible. It has been proved by other authors (Adamson et al., 1988), that severe injury and retarded repair of
epithelium disturbs normal epithelial-fibroblast interaction and is sufficient to promote the fibrotic process. Less
severe injury involving the endothelium only is not associated with fibrosis.
Lungs congestion (Hyperaemia) represents the increase of blood in a territory, due to dilatation of small vessels.
According to the mechanism, it may be active or passive. It divided into active and passive hyperaemia. Active
hyperaemia congestion is a result of arteriolar distension (e.g., skeletal muscle activity, inflammation, local neuron
vegetative reaction). Passive hyperaemia congestion, also termed stasis, is a consequence of an impaired venous
drainage (heart failure, compression or obstruction of veins), followed by dilatation of veinules and capillaries
(Aster et al., 2009). Alveolar walls are thickened due to dilated capillaries. Alveolar lumens are filled
with transudate (amorphous, eosinophilic and homogenous) which replaced the air, red blood cells
(microhemorrhages) and hemosiderin-laden macrophages (also called heart failure cells).
With progression, interstitial fibrosis may appear and, together with hemosiderin pigmentation, generates the
aspect of "brown induration". Extensive fibrosis leads to intrapulmonary hypertension. Passive congestion of the
lung, hemosiderin-laden macrophages contain in cytoplasm hemosiderin pigment (brown, granular), resulted from
destruction of red blood cells in alveolar lumen (Knudsen and Ochs 2018).
In the current study, tramadol showed marked degenerative changes of bronchiolar epithelium cells. The alveolar
septal walls were thickened with cellular changes and capillary congestion. The basement membranes showed
marked thickening and the airway lumens contained abundant mucinous secretions. Previously, similar
ultrastructural airway changes were detected in chronic and acute tramadol tratment. The numbers of Clara cells
and ciliated epithelial cells decreased while the numbers of goblet cells increased with numerous secretory
granules containing mucus.
The basement membrane was thickened due to deposition of collagen. The myofibroblast sheath was also
thickened due to high content of collagen and myofibroblasts in addition to smooth muscle hypertrophy and
hyperplasia. The bronchial epithelium was shrunken with pyknotic nuclei. Moreover, there were platelet

activation and inflammatory cell infiltration in the perivascular and peribronchiolar areas. In the airway smooth
muscle, the mitohcondria increased in number with ultrastructural changes this results agreed with Hussam et
al.,(2017).They suggested that tramadol toxic substance metabolites effects on lungs tissue causes the previous
diagnosis.
2-Histochemistry findings of total proteins
In the present study, the daily administration of rats with the therapeutic dose of tramadol (20, 40 & 80 mg/kg b.w.)
caused a remarkable reduction in the total proteins contents in lungs cells of treated rats in comparison to control
rats. Treatment of rats with tramadol for 30 days induced a marked decrease in the protein content in lungs cells.
More reduction in proteins was manifested in the cells treated for 3 monthes, where the proteinic granules were
clearly reduced in amount and stainability. The reduction of protein contents observed in this study may be
attributed damaged of kidney cells by tramadol toxic substance.
In addition, Palla et al., (1987), postulated that in many kidney diseases, the permeability of the glomerular
capillaries is increased leading to increased levels of excreted proteins. They added that any lesions produced in the
kidney tubules will eventually cause dysfunction in the transport mechanism to and from the renal epithelium.
3- Histochemical results of polysaccharides
In the present study, obvious alteration in the histochemical results of lungs cells of the rats treated with 40 mg/kg
body weight of tramadol was noted. Carbohydrate was found to undergo a remarkable diminution in all treated
groups in comparison to the lungs of the control rats. Such diminution exhibited time dependent characteristics.
However, such presently decrease in carbohydrate content can be explained by (Chen et al., 1999) who stated that
initiation of lipid peroxidation, necrosis and subsequent impairment in cellular metabolism collectively altered the
major cellular components, including protein, and glycogen. In general, the reduction of carbohydrates components
under the effect of tramadol could be due to the release of hydrolytic enzymes from the ruptured lysosomes under
the toxic effect of the toxic agents (Shalaby, 1985). The above detected depletion in glycogen inclusions supported
by previous findings postulated by Popp and Cattely, (1991) that indicated that glycogen accumulation may be
decreased as manifestation of toxicity, which is apparently due to impairment of enzymatic activity for glycogen
catabolism or decrease in glycogen synthesis. The data collected from the present investigation could suggest that
depletion of lungs glycogen which takes place under such conditions might be attributed to the effect of tramadol
on glucose absorption or on the enzymes involved in the process of glycogenesis or/and glycolysis (Jarrar & Taib,
2012). Elyazji et al. (2013) reported that there is a general increase in serum glucose levels in rats in response to
tramadol administration. Hepatocytes of the periportal zones were more affected than the perivenous hepatocytes
which might indicate glycogenesis was more affected than glycolysis in the periportal hepatocytes which is
metabolized by the perivenous cells that contain higher levels of glucokinase and pyruvate kinase during the post
absorptive phase. The heterogeneous reduction in glycogen content between the same types of cells may indicate a
difference in the overall release of glucose. Hepatocytes in the area surrounding the terminal afferent are mainly
gluconeogenic, while those ones surrounding the terminal efferent venules are mainly glycolytic and lipolytic and

are involved in biotransformation as general detoxification mechanism such mode of occurrence of these inclusions
supported by the findings of (Jarrar & Taib, 2012).

On the contrary, a good support is provided to the present results. In this regard, Abdel-Raheem et al. (1991)
indicated that marked declines occurred in the liver, kidney and brain glycogen contents as consequences of
administration of heroin at fixed doses to adult albino rats. In addition, Zahran, (1994) found that heroin
administration led to a duration and dose-dependent decrease of glycogen content in liver and kidney of rats.
Such decrease was found to be also concomitant with marked hepatic and renal G-6-Pase declines. The possible
interpretation of our results of carbohydrates depletion in the present study could be attributed to the toxication
effects of tramadol on the testies cells; under pathological condition the cells lost their capacity to metabolize
glycogen normally.
The results showed that treated rats with tramadol caused depletion of carbohydrates in the cytoplasm of renal
tubules. This result was in correspondence with other studies reported by Sakr et al,. (2003) due to the treatment of
gibberellin to the rats, and Elyazji et al. (2013) due to the use of a variety of animals under different pathological
conditions.

4-Histochemical observations for collagen fibers
IN the current studies increase of connective tissue fibres noticed by Mallory terrible stain in tramadol-treated
group appeared as large abounded of fibrosis around lungs cells.
Lungs fibrosis may be due to generation of free radicals inducing oxidative stress leads to molecular and cellular
damage which are considered the cause of tramadol toxic effects on the different body organs (Argani et al., 2011).
The effects of released reactive oxygen species (ROS) by normal respiratory system are counteracted by
glutathione and antioxidants enzymes such as catalase and peroxidase; therefore more generation of ROS via
tramadol toxic substance leads to the balance disturbance with antioxidants defense mechanism inducing toxic
cellular substances which lead to histopathological changes (Lee, 2010).

5-Ultrastructural lesions
The lung is the essential organ of respiration and the organ that receives the entire cardiac output. Also, the lung
plays an important role in host defense and regulation of circulating levels of biologically active materials by
extensive surface of pulmonary vascular bed. The study of the relation between tramadol toxic substance and
respiratory health needs to take into account the anatomical, histological and the toxicological effect. Tramadol also
have various harms on many systems, not include respiratory system only but also exerted on variety of organs of
living bodies such as testis, brain, kidney and liver.
Acute lung injury is a common clinical illness. The current acute lung injury mortality rate is as high as 35%-40%
and reaches to 50% in acute respiratory distress syndrome (Wheeler et al., 2007). Acute lung injury pathogenesis is
complex, and there is still much controversy to be had before we reach a definitive conclusion (Villar et al., 2011).
Increased oxidative stress has been implicated in its pathogenesis (Choi et al., 2012). Tramadol induced acute lung
injury in rat model is a classic animal model. . In recent years, some researchers found that mitochondrial
dysfunction plays an important role in the course of acute lung injury.
.

Mitochondria utilize approximately 98% of total body oxygen consumption. This would maintain tissue oxygen
levels by decreasing demand and protect against cell death.So mitochondrial dysfunction is the key factor to cell
damage. In sepsis, mitochondrial dysfunction in vital organs can make cell organisms lack energy, causing multiple
organ failure. The lung is a special organ relatively susceptible to injury. The mitochondrion is a complex and
sensitive organelle Acute lung injury can lead to abnormal mitochondrial structure and function and tends to cause
abnormal mitochondria organelles or other changes in the entire cell, thereby increasing the degree of acute lung
injury meanwhile, mitochondrial dysfunction is also prone to result in acute lung injury (Singer, 2014).In lung
tissue treatment with tramadol can produce large radical NO, O-2
, and ONOO-
. The mitochondrial film, which is
rich in unsaturated fatty acids, is a major free radical attack target. These lead to mitochondrial membrane swelling
expansion, lipid oxidation increase, and decrease membrane fluidity.
Mitochondrial ATP enzyme activity and mitochondrial ATP production were also decreased because of these.
Mitochondria have intrinsic defense mechanisms to protect against ROS-induced damage through its large array of
antioxidants (e.g., superoxide dismutase, glutathione, thioredoxin) (Yin et al., 2012).

The formation of various density-graded granules in the cytoplasm of lung tissues and an increased number of
vacuoles were observed both in group IIIc. These results agreed with Ji-Young et al., (2012) which explain the
autophagy pathway is a catabolic intracellular process that activates the lysosomal degradation pathway. During
autophagy, cytoplasmic structures are sequestered into double-membraned or multi-layered autophagosomes and
fused with lysosomes to form secondary lysosomes or autophagolysosomes for degradation.
In addition to the formation of various density-graded granules in the cytoplasm of lung tissues, an increased
number of vacuoles were observed in treated groups with tramadol and combination control groups. Moreover,
the autophagy pathway is a catabolic intracellular process that activates the lysosomal degradation pathway.
Most of the published articles focused on a fact which shows that generation of free radicals inducing oxidative
stress leads to molecular and cellular damage which are considered the cause of tramadol toxic effects on the
different body organs (Argani et al., 2011).
The effects of released reactive oxygen species (ROS) by normal respiratory system are counteracted by
glutathione and antioxidants enzymes such as catalase and peroxidase; therefore more generation of ROS via
tramadol toxic substance leads to the balance disturbance with antioxidants defense mechanism inducing toxic
cellular substances which lead to histopathological changes (Lee, 2010).
The present study showed that tramadol increases fibrous tissues formation in the lung interstitial tissues and
around alveoli depending on its dose in consistency with Katrin et al.,(1986) who referred to the fact that tramadol
can be trigger to stimulate fibroblast proliferation via mediators which are induced by the epithelial cells of airway
passages.

According to Esposito et al., (2000) there is a correlation between tramadol cytotoxicity and mitochondrial enzyme
activity disturbance and its ability to react with the nucleus receptors to prevent genetic transcription of proteins
that are secreted by fibroblasts, macrophages, monocytes, and endothelial cells. Therefore, cyclosporine affects the

cell of lung tissues as a result of multiple effects such as carbohydrates depletion in the cytoplasm of lung cell that
leads to the lung structure disturbance which is supported in the current study by a negative stain of Periodic
Acid-Schiff.

Most recently, Minisy F. et al., (2020) investigated that chronic exposure to tramadol induces testicular damage in
adult and adolescent rats. Histological and ultrastructural examinations revealed that tramadol induced hemorrhage
of blood vessels, intercellular spaces, interstitial vacuoles, exfoliation of germ cells in lumen, cell apoptosis,
chromatin degeneration of elongated spermatids, and malformation of sperm axonemes. Moreover, tramadol
disrupted collagen metabolism and cell cycle progression. In conclusion, although tramadol is very commonly
prescribed, it should be administered with the consideration of the risk to benefit ratio. Tramadol is one of the most
common causes of poisoning in adult male patients with the previous history of drug addiction and psychological
problems and suicide is the most common motivation for its use in this group of the patients.
Declarations
Source of Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit
sectors.
Competing Interests Statement
The authors declare no competing financial, professional and personal interests.
Consent for publication
We declare that we consented for the publication of this research work.
References
1. Abbas RA, Hammam RAM, El-Gohary SS, Sabik LME, Hunter MS. Screening for common mental disorders
and substance abuse among temporary hired cleaners in Egyptian governmental hospitals, Zagazig city, Sharqiag
governorate. Int J Occup Environ Med 2013;4:13–26.
2. Abdel-Raheem, K., El-Mossallamy, N., El-adeki, M., El-Gabry, M., Elamany,N.,(1991): Carbohydrate
metabolism in response to administration of heroin at fixed doses. Proc. Zool. Soc. A.R. Egypt 22, 149–165.
3. Abolmaged S, Kodera A, Okasha T, Gawad T, Rawson R. Tramadol .Tramadol use in Egypt: emergence of a
major new public health problem.Can J Addiction Med 2013;4:5.
4. Adamson I.Y.R. , Young L. and Bowden D.H. (1988). Relationship of alveolar epithelial injury and repair to the
induction of pulmonary fibrosis. Am. J. Pathol. 130,377-383.
5. Adamson IYR: Alveolar Injury and Repair. Electron Microscopy of the Lung. Volume 48. Edited by:
Schraufnagel D, Dekker SD. 1990, New York: Marcel Dekker; Lenfant C (Series Editor): Lung Biology in Health
and disease, 165-172.
6. Agusti AG, Noguera A, Sauleda J, Sala E, Pons J, Busquets X. Systemic effects of chronic obstructive
pulmonary disease. Eur Respir J. 2003;21:347–60.
7. Anderson LJ, Freeman G. Effects of NO2 on the lungs of aging rats. Exp Mol Pathol. 1977;27, 353 – 365.
8. Argani H., Ghorbanihaghjo A., Rashtchizadeh N., Seifirad S., and Rahbarfar Y., “Effect of cyclosporine-a on
paraoxonase activity in wistar rats,” International Journal of Organ Transplantation Medicine, vol. 2, no. 1, pp. 25–
31, 2011.

9. Aster J, Vinay Kumar, Abul K. Abbas; Nelson Fausto (2009). Robbins & Cotran Pathologic Basis of Disease
(8th ed.). Philadelphia: Saunders. p. 113. ISBN 978-1-4160-3121-5.
10. Barkin RL, (2008). Extended-release tramadol (ULTRAM ER):a pharmacokinetic, and pharmacodynamics
focus on effectiveness and safety in patients with chronic/persistent pain. Am. J. Ther., 15 (2): 157-166.
11. Barreiro E, del Puerto-Nevado L, Puig-Vilanova E, Perez-Rial S, Sanchez F, Martinez-Galan L, Rivera S, Gea
J, Gonzalez-Mangado N, Peces-Barba G. Cigarette smoke-induced oxidative stress in skeletal muscles of mice.
Respir Physiol Neurobiol. 2012;182:9–17.
12. Bita D, Anahita A.G. and Fares N. Pathological effects of tramadol on lung tissue in the cadaver referred to
legal medicine organization of Tehran 2008-2013 international journal of recent scientific Research Vol. 6, Issue,
12, pp. 8026-8029, December, 2015.
13. Boysen M, Zadig E, Digernes V, et al.Intracytoplasmic lumina with and without cilia in both normal and
pathologically altered nasal mucosa. Ultrastr Pathol .1980;1, 477- 485.
14. Calfee CS, Matthay MA: Nonventilatory treatments for acute lung injury and ARDS. Chest. 2007, 131:
913-920. 10.1378/chest.06-1743.
15. Carr R, Lucas S, Liomba N, Whitten R, Grau G, Mackenzie C, Molyneux M, Taylor T, Das A: Lung pathology
in fatal pediatric malaria. Am J Trop Med Hyg. 1999, 61 (Supplement 1): 260-270.
16. Chen, W., Shockcor, J.P., Tonger, R., Hunter, A., Gartner, C. and Nelson,S.D., (1999): Protein and nonprotein
cysteinylthiol modification by N-acetylp benzoquinoneimine via a novel ipso adduct. Biochemistry, 38: 8159.
17. Choi JS, Lee HS, Seo KH, Na JO, Kim YH, Uh ST, Park CS, Oh MH, Lee SH, Kim YT: The effect of
post-treatment N-acetylcysteine in LPS-induced acute lung injury of rats. Tuberc Respir Dis (Seoul). 2012, 73:
22-31. 10.4046/trd.2012.73.1.22.
18. Chua CLL, Brown G, Hamilton JA, Rogerson S, Boeuf P: Monocytes and macrophages in malaria: protection
or pathology?. Trends Parasitol. 2013, 29: 26-34.
19. Chyczewska E. , Chyczewski L., Bankowski E., Sulkowski S. And Niklinski J. (1993). Stimulation of alveolar
macrophages by BCG vaccine enhances the process of lung fibrosis induced by bleomycin.Folia Histochem.
Cytobiol. 31, 113-116.
20. Cox G, Crossley J, Xing Z. Macrophage engulfment of apoptotic neutrophils contributes to the resolution of
acute pulmonary inflammation in vivo. Am J Respir Cell Mol Biol 1995;12:232–237.
21. Cottrell, T. S., Levine, 0. R., Senior, R. M., Wiener, J., Spiro, D., and Fishman, A. P. (1967). Electron
microscopic alterations at the alveolar level in pulmonary edema. Circulat. Res., 21, 783-797.
22. Darby, I.A.; Zakuan, N.; Billet, F.; Desmoulière, A. The myofibroblast, a key cell in normal and pathological
tissue repair. Cell. Mol. Life Sci. 2016, 73, 1145–1157.
23. Davarian A, Fazeli SA, Azarhoush R, et al.Histopathologic changes of rat tracheal mucosa following
formaldehyde exposure.Inter J Morphol. 2005;23: 369-372.
24. Domenici L, Pieri L, Galle MB, Romagnoli P, Adembri C. Evolution of endotoxin-induced lung injury in the
rat beyond the acute phase. Pathobiology 2004;71:59–69.

25. Elyazji NR, Abdel-Aziz I, Aldalou A and Shahwan O. 2013. The effects of tramadol hydrochloride
administration on the hematological and biochemical profiles of domestic male rabbits. IUG J Natural and Eng
Studies,21:51-65.
26. Erjefalt JS, Uller L, Malm-Erjefalt M, Persson CG. Rapid and efficient clearance of airway tissue granulocytes
through transepithelial migration. Thorax 2004;59:136–143.
27. Esposito C., Fornoni A., Cornacchia F., “Cyclosporine induces different responses in human epithelial,
endothelial and fibroblast cell cultures,” Kidney International, vol. 58, no. 1, pp. 123–130, 2000.
28. Faria J, Barbosa J, Leal S, Afonso LP, Lobo J, Moreira R, Queiros O,Carvalho F, Dinis-Oliveira RJ (2017).
Effective analgesic doses oftramadol or tapentadol induce brain, lung and heart toxicity inWistar rats. Toxicology
385:38–47.
29. Finecold, M. J.: Interstitial pulmonary edema.An electron microscopic study of the pathology of
staphylococcal enterotoxemia in rhesus monkeys. Lab. Invest. 16: 912, 1967.
30. Gaspani L, Bianchi M, Limiroli E, Sacerdote P (2002). The analgesic drug tramadol prevents the effect of
surgery on natural killer cell activity and metastatic colonization in rats. J Neuroimmunol, 129 (1-2),18‐24.
31. Gil, J.; Martinez-Hernandez, A. The connective tissue of the rat lung: Electron immunohistochemical
studies.J. Histochem. Cytochem. 1984, 32, 230–238.
32. Greene, D. G.: Pulmonary edema. In Handbook of Physiology, sec. 3, vol. II, edited by W. O.Fenn and H.
Rahn. Washington, D. C., American Physiological Society, 1965, p. 1588.
33. Grond S, Sablotzki A (2004). Clinical pharmacology of tramadol. Clin Pharmacokinet 43(13): 879-923.
34. Hamdi E, Gawad T, Khoweiled A, Sidrak AE, Amer D,Mamdouh R, Fathi H, Loza N. Lifetime prevalence of
alcohol and substance abuse in Egypt: a community survey. Substance Abuse 2013; 34:97–114.
35. Harris CC, Kaufman DG, Jacksons F, et al.Atypical cilia in the tracheobronchial epithelium of the hamster
during respiratory carcinogenesis. J Pathol. 1974; 114, 17 - 19.
36. Haslett C. Granulocyte apoptosis and its role in the resolution and control of lung inflammation. Am J Respir
Crit Care Med 1999;160:S5–S11.
37. Hee L, Dinudom A, Mitchell AJ, Grau GE, Cook DI, Hunt NH, Ball HJ: Reduced activity of the epithelial
sodium channel in malaria-induced pulmonary oedema in mice. Int J Parasitol. 2011, 41: 81-88.
10.1016/j.ijpara.2010.07.013.
38. Hotchkiss, R.D. (1948): A Microchemical Reaction Resulting in the Staining of Polysaccharide Structures in
Fixed Tissue Preparations. Archives of iochemistry, 16, 131.
39. Hussam AS,Hamed S H, Misbahuddin M R, Mansour I S, Amer S A and Mohamad-Nidal A K
(2017).Therapeutic effects of co-inhaled roflumilast or formoterol and fluticasone on asthma-induced
ultrastructural changes in murine airways. Tropical Journal of Pharmaceutical Research November 2017; 16 (11):
2637-2644.
40. Jarernsiripornkul N, Krska J, Richards RM, Capps PA (2003). Patient reporting of adverse drug reactions
:useful information for pain management? Eur J Pain 7(3): 219-224.

41. Jarrar B. and Taib N. (2012): Histological and histochemical alterations in the liver induced by lead chronic
toxicity. Saudi Journal of Biological Sciences; 19(2): 203–210.
42. Javedan M, Entezarizaher T. Cytotoxic effect of formaldehyde vapour on rat nasal mucosa during 3- and 30-
day periods. J Qazv Med Scien Univ. 1999; 2, 17-23.
43. Ji-Young SHIN1, Hwang-Tae LIM1,2, Arash MINAI-TEHRANI1, Mi-Suk NOH1,2, Ji-Eun KIM1, 2,Ji-Hye
KIM1,2, Hu-Lin JIANG1, Rohidas AROTE6, Doo-Yeol KIM7, Chanhee CHAE7, Kee-Ho LEE8,Mi-Sook
KIM8and Myung-Haing CHO1,2,3,4,5,(2012). Aerosol delivery of beclin1 enhanced the anti-tumor effect
ofradiation in the lungs of K-rasLA1mice. Journal of Radiation Research, 2012, 53, 506–515.
44. Kaewamatawong, T.; Kawamura, N.; Okajima, M.; Sawada, M.; Morita, T.; Shimada, A. Acute pulmonary
toxicity caused by exposure to colloidal silica: Particle size dependent pathological changes in mice.Toxicol.
Pathol. 2005, 33, 745–751.
45. Katrin E., Michael R., Janette K., Peter R., and Glanville A.R., Cyclosporine A mediates fibroproliferation
through epithelial cell,” Transplantation, vol. 77, no. 12, pp. 1886–1893, 2004.
46. Kelley J. (1990). Cytokines of the lung. Am. Rev. Respir. Dis. 141, 765-788.
47. Kimbell J, Overton J, Subramaniam R, et al.Dosimetry modeling of inhaled formaldehyde : Binning nasal flux
predictions for quantitative risk assessment.Toxicol Scienc. 2001;64, 111–121 .
48. Knudsen, L., and Ochs, M. (2018). The micromechanics of lung alveoli: structure and function of surfactant
and tissue components. Histochem. Cell Biol. 150, 661–676. doi: 10.1007/s00418-018-1747-9
49. Kovach MA, Standiford TJ. The function of neutrophils in sepsis. Curr Opin Infect Dis 2012; 25: 321–327.
50. Krichesky B., (2009) : A Modification of Mallory's Triple Stain. Pages 97-98 | Published online: 12 Jul 2009.
Journal stain technology , volume 6 1931- issue 3.
51. Lee C.R., McTavish D. and Sorkin E.M.(1993). Tramadol: a preliminary review of its pharmacodynamic and
pharmacokinetic properties and therapeutic potential in acute and chronic pain states, Drugs, Vol 46: 313-340.
52. Lee J, Taneja V, Vassallo R. Cigarette smoking and inflammation: cellular and molecular mechanisms. J Dent
Res. 2012;91:142–9.
53. Lee J., “Use of antioxidants to prevent cyclosporine a toxicity,” Toxicological Research, vol. 26, no. 3, pp.
163–170, 2010.
54. Lillie R.D. (1954): Histological techniques and practical histochemistry McGraw-Hill, U.S.A.
55. Liu CY, Liu YH, Lin SM, et al. Apoptotic neutrophils undergoing secondary necrosis induce human lung
epithelial cell detachment. J Biomed Sci 2003;10:746–756.
56. Luft, J. H.: Ultrastructural basis of capillary permeability. In The Inflammatory Process,edited by B. W.
Zweifach, L. Grant, and R. T. McCluskey. New York, Academic Press,1965, p. 148.
57. Martin TR, Nakamura M, Matute-Bello G. The role of apoptosis in acute lung injury. Crit Care Med 2003; 31:
184–188.
58. Matthiesen T., Wohrmann T., Coogan T.P., and Uragg H. The experimental toxicology of tramadol: an
overview in toxicology book.2010,p;407-20.

59. Mazia , D.; Bewer P.A.; and Affest M.; (1953): The cytochemical staining and measurements of protein with
mercuric bromophenol blue. Biol Bull 104,57-67.
60. Medan D, Wang L, Yang X, Dokka S, Castranova V, Rojanasakul Y. Induction of neutrophil apoptosis and
secondary necrosis during endotoxin-induced pulmonary inflammation in mice. J Cell Physiol 2002;191:320–326.
61. Minisy F , Hossam H. Shawki , Abdelfatteh El Omri,4 Ahmed A. Massoud, Enayat A. Omara, Fatma G.
Metwally, Manal A. Badawy, Neveen A. Hassan, Nabila S. Hassan, & Hisashi Oishi (2020). Pomegranate Seeds
Extract Possesses a Protective Effect against Tramadol-Induced Testicular Toxicity in Experimental Rats. BioMed
Research International. Article ID 2732958, 12 pages https://doi.org/10.1155/2020/2732958.
62. Monticello TM, Swenberg JA, Gross EA, et al.Correlation of regional and nonlinear formaldehyde-induced
nasal cancer with proliferating populations of cells. Cancer Res. 1996; 56, 1012–1022.
63. Naota, M.; Mukaiyama, T.; Shimada, A.; Yoshida, A.; Okajima, M.; Morita, T.; Inoue, K.; Takano,
H.Pathological study of acute pulmonary toxicity induced by intratracheally instilled Asian sand dust
(Kosa).Toxicol. Pathol. 2010, 38, 1099–1110.
64. Naya M, Nakanish J. Risk assessment of formaldehyde for the general population in Japan Biochem Biophys
Res Commun.2005; 295, 663–667.
65. Neelam B, Uppal V, Pathak D. Toxic effect of formaldehyde on the respiratory organs of rabbits: A light and
electron microscopic study. Toxicol Indust Health. 2011;27(6) 563–569.
66. Njoya HK, Ofusori DA, Nwangwu SC, et al.Histopathological effect of exposure of formaldehyde vapour on
the trachea and lungs of adult wistar rats. Intern Integr Biol.2009; 7, 160-165.
67. Ohashi Y, Nakai Y, Ikeoka H. Colchicine induced ciliated vacuoles in tracheal mucosa . Acta Otolaryngol
(Stockh) suppl.1991;486, 64-271.
68. Ohtsuka R et al. A further comparative study on early histological changes in respiratory tract of Brown
Norway and Fischer-344 rats after shortterm inhalation of formaldehyde aerosol. J. T. Pathol. 1998; 11:235-40.
69. Ojha R. and Bhatia SC.(2010).Tramadol dependence in a patient with no previous substance history. Prim
Care Companion J Clin Psychiatry 12(1): CC.
70. Oliveira MV, Abreu SC, Padilha GA, Rocha NN, Maia LA, Takiya CM, Xisto DG, Suki B, Silva PL, Rocco
PR. Characterization of a Mouse Model of Emphysema Induced by Multiple Instillations of Low-Dose Elastase.
Front Physiol. 2016;7:457.
71. Palla,R.; Patrenos Terg G.; Galigane, R; Brtell, A.; Romono, M.;Alessandr, M. and Bartalla, A. (1987):
Comparative effects of gentamicin,amikacin and dactimicin on excretion of acety1 beta-d-glucosamidase (Nag)
and kidney histological pattern.
72. Perl M, Lomas-Neira J, Venet F, Chung CS, Ayala A. Pathogenesis of indirect (secondary) acute lung injury.
Expert Rev Respir Med 2011; 5: 115–126, doi: 10.1586/ers.10.92.
73. Pforte A., Gerth C., Voss A., Beer B., Haussinger K., Jutting U., Burger G. and Ziegler-Heitbrock H.W.L.
(1993). Proliferating alveolar macrophages in BAL and lung function changes in interstitial lung disease. Eur.
Respir. J. 6, 951 -955.

74. Popp, J.A. AND Cattely, R.C., (1991): Hepatobillary system. In: Handbook of toxicologic and pathology.
(eds. W.M. Haschek and C.G. Rousseaux).Academic Press Inc., Diego, pp. 269-314.
75. Porter, D.W.; Millecchia, L.L.; Willard, P.; Robinson, V.A.; Ramsey, D.; McLaurin, J.; Khan, A.; Brumbaugh,
K.;Beighley, C.M.; Teass, A.; et al. Nitric oxide and reactive oxygen species production causes progressive damage
in rats after cessation of silica inhalation. Toxicol. Sci. 2006, 90, 188–197.
76. Pryhuber, G.S.; Huyck, H.L.; Baggs, R.; Oberdörster, G.; Finkelstein, J.N. Induction of chemokines by
low-dose intratracheal silica is reduced in TNF I (p55) null mice. Toxicol. Sci. 2003, 72, 150–157.
77. Robertson, B., Enhorning, G., Ivemark, B., Malmqvist, E., and Mod6e,J. (1971). Experimental respiratory
distress induced by paraquat. J. Path., 103, 239-244.
78. Rocha, N.d., de Oliveira, M.V., Braga, C.L. et al., (2017). : Ghrelin therapy improves lung and cardiovascular
function in experimental emphysema. Respir Res 18, 185 https://doi.org/10.1186/s12931-017-0668-9.
79. Rydell-Törmänen K., Uller l., Erjefält J.S.European Respiratory Journal 2006 28: 268-274.Ryoko K, Izumi K,
Masanori T, Mio M., et al.Alteration in airway microvascular leakage induced by sensorineural stimulation in rats
exposed to inhaled formaldehyde. Toxicol Letters. 2010;199, (3) 254-260.
81. Said, S. I., Longacher, J. W., Davis, R. K.,Banehjee, C. M., Davis, W. M., AND Wooddell,W. J.: Pulmonary
gas exchange during induction of pulmonary edema in anesthetized dogs. J. Appl. Physio!. 19: 403, 1964.
82. Sakr , SA. El-Messedy, FA and Abdel-Samei Ha. (2003): Histopathological and histochemicals effects of
gibberellin A3 on the kidney of albino rats. J Egypt Grrm Soc Zool 38.1-10.
83. Samaka R, Girgis N, Shams T (2012). Acute toxicity and dependence of tramadol in albino rats: relationship of
nestin and notch 1 as stemcell markers. J Am Sci 8(6):313–327.
84. Senaldi G, Vesin C, Chang R, Grau GE, Piguet PF: Role of polymorphonuclear neutrophil leukocytes and their
integrin CD11a (LFA-1) in the pathogenesis of severe murine malaria. Infect Immun. 1994, 62: 1144-1149.
85. Shipton E.A.Tramadol – present and future, Anaesth. Intensive Care, Vol 28: 363-374, 2000.
86. Sibille Y. and Reynolds H.Y. (1990). Macrophages and polymorphonuclear neutrophils in lung defense and
injury. Am. Rev. Respir.Dis. 141,471-501.
87. Singer M: The role of mitochondrial dysfunction in sepsis-induced multi-organ failure. Virulence. 2014, 5:
66-72. 10.4161/viru.26907.
88. Soueif MI, Youssuf GS, Taha HS, Moneim HA, Sree OA, Badr KA,Salakawi M, Younes FA. Use of
psychoactive substances among secondary school students in Egypt: a study on a nationwide representative sample.
Drug Alcohol Depend 1990;26:63–79.
89. Soueif MI,Yunis FA,Taha HS.Extent and patterns of drug abuse and its associated factors in Egypt. Bull Narc
1986;38 :113–120.
90. Staub, N. C, Nagano, H., AND Pearce, M. L.:Pulmonary edema in dogs, especially the sequence of fluid
accumulation in lungs. J.Appl. Physiol. 22: 227, 1967.
91. Suki B, Lutchen KR, Ingenito EP. On the progressive nature of emphysema: roles of proteases, inflammation,
and mechanical forces. Am J Respir Crit Care Med. 2003;168:516–21.

92. Takhtfooladi, M. A., Jahanshahi, A., Sotoudeh, A., Takhtfooladi, H. A., & Aslani, K. (2013). Effect of
tramadol on lung injury induced by skeletal muscle ischemia-reperfusion: an experimental study. Jornal brasileiro
de pneumologia : publicacao oficial da Sociedade Brasileira de Pneumologia e Tisilogia, 39 (4), 434 – 439.
93. Tanaka, T.; Kishimoto, T. The biology and medical implications of interleukin-6. Cancer Immunol. Res.
2014,2, 288–294.
94. Tandler B, Liedtke CM. Tunnels in the lining epithelium rabbit trachea. Anat Rec .1981;199: 252.
95. Tandler B, Sherman JM, Boat TF. Surface architecture of the mucosal epithelium of the cat trachea
Cartilaginous portion. Am J Anat. 1983 I;168, 119-131.
96. Tarkkila P., M. Tuominen and L. Lindgren (1998): Comparison of respiratory effects of tramadol and
pethidine. Eur J. Anaesthesiol., 15 (1):64-8.
97. Tavassoli N, Lapeyre-Mestre M, Sommet A, Montastruc JL (2009). Reporting rate of adverse drug reactions to
the French pharmacovigilance system with three step 2 analgesic drugs: dextropropoxyphene ,tramadol and
codeine (in combination with paracetamol). Br J Clin Pharmacol 68(3): 422-426.
98. Torikata C, Tacheuchi H, Yamaguchi H, et al.Abnormal cilia in bronchial mucosa: Case reports of non
smoking women with bronchogenic carcinomas and an experimental model in guinea pigs.Virchows Arch [Pathol
Anat]1976; 371, 121 – 129 (cited by Tandler et al 1983 .
99. Uhley, H., Leeds, S. E., Sampson, J. J., AND Friedman, M.: Some observations on the role of lymphatics in
experimental acute pulmonary edema. Circulation Res. 9: 688, 1961.
100.Valérie L, Matthieu A, Sarah A, et al. MAPKand PKC/CREB-dependent induction of interleukin-11 by the
environmental contaminant formaldehyde in human bronchial epithelial cells. Toxicol. 2012;292 (12) 13– 22.
101.Villar J, Blanco J, Añón JM, Santos-Bouza A, Blanch L, Ambrós A, Mosteiro F, Basaldúa S, Fernández RL,
Kacmarek RM, ALIEN Network: The ALIEN study: incidence and outcome of acute respiratory distress syndrome
in the era of lung protective ventilation. Intensive Care Med. 2011, 37: 1932-1941.
102.Vogelmeier CF, Criner GJ, Martinez FJ, Anzueto A, Barnes PJ, Bourbeau J, Celli BR, Chen R, Decramer M,
Fabbri LM, et al. Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Lung
Disease 2017 Report: GOLD Executive Summary. Eur Respir J. 2017:49.
103.Wajant, H.; Pfizenmaier, K.; Scheurich, P. Tumor necrosis factor signaling. Cell Death Differ. 2003, 10, 45–
65.
104.Wang S.Q., Li C.S. and Song Y.G (2009). Multiply organ dysfunction syndrome due to tramadol intoxication
alone. Am J Emerg Med 27(7): 903-907.
105.Warren PM, Taylor JH, Nicholson KE, Wraith PK, Drummond GB (2000). Influence of tramadol on the
ventilatory response to hypoxia in humans. Br J Anaesth 85(2): 211-216.
106.Wheeler AP, Bernard GR: Acute lung injury and acute respiratory distress syndrome: a clinical review.
Lancet. 2007, 369: 1553-1564. 10.1016/S0140-6736(07)60604-7.
107.Yin F, Sancheti H, Cadenas E: Mitochondrial thiols in the regulation of cell death pathways. Antioxid Redox
Signal. 2012, 17: 1714-1727. 10.1089/ars.2012.4639.

108.Zahran, M.F.,( 1994): Effect of heroin administration on certain biochemical aspects in adult rabbits. Egypt. J.
Med. Sci. 15 (1), 69–78.
109.Zarnescu, O.; Brehar,F.M.;Chivu,M.andCiurea, A.V. (2008): Immunohistochemical localization of
cas-pase-3, caspase-9 and Bax in U87 glioblasto maxenografts. J Mol Histol., 39: 561-569.
110.Zeidler, P.; Hubbs, A.; Battelli, L.; Castranova, V. Role of inducible nitric oxide synthase-derived nitric oxide
in silica-induced pulmonary inflammation and fibrosis. J. Toxicol. Environ. Health A 2004, 67, 1001–1026.

Acute Abuse of Tramadol Induces Intensive Pulmonary Histopathological Trauma in Rats - An Experimental Comprehensive Overview

Recommended

Recommended

More Related Content

What's hot

What's hot (9)

Similar to Acute Abuse of Tramadol Induces Intensive Pulmonary Histopathological Trauma in Rats - An Experimental Comprehensive Overview

Similar to Acute Abuse of Tramadol Induces Intensive Pulmonary Histopathological Trauma in Rats - An Experimental Comprehensive Overview (20)

More from Associate Professor in VSB Coimbatore

More from Associate Professor in VSB Coimbatore (20)

Recently uploaded

Recently uploaded (20)

Acute Abuse of Tramadol Induces Intensive Pulmonary Histopathological Trauma in Rats - An Experimental Comprehensive Overview