This study introduces a new quantitative analysis technique for evaluating immunohistochemistry images of the spinal dorsal horn (SDH) in a rat model of chronic neuropathic pain. Eight rats underwent a spinal cord dorsal hemisection injury to induce neuropathic pain. Immunofluorescent staining for NeuN and GABA was performed on spinal cord sections. A novel ellipse region of interest was defined based on the distribution of NeuN-positive neurons to standardize measurements between samples. This technique showed a significant loss of GABAergic neurons and decrease in GABA immunoreactivity in deeper regions of the injured SDH compared to the uninjured side. This method allows for quantitative comparison of cell distributions and immunoreactivity intensities in the SDH.

1. 8
Analytical and Quantitative Cytopathology and Histopathology®
0884-6812/21/4301-0008/$18.00/0 © Science Printers and Publishers, Inc.
Analytical and Quantitative Cytopathology and Histopathology®
OBJECTIVE: The quantitative analysis of the changes
of the in situ GABA-immunoreactivity in the dorsal
horn of the spinal cord (SDH) remains challenging.
Therefore, we introduced a novel evaluation technique
based on immunohistochemistry images.
STUDY DESIGN: Eight Sprague-Dawley rats under-
went a spinal cord dorsal hemisection, and we induced
chronic neuropathic pain (CNPSCI). Immunofluorescent
staining with markers of NeuN and GABA was per-
formed. We developed a method with an ellipse region
of interest (ROI) and baselines according to the dis-
tribution of NeuN-positive neurons. We investigated
the distribution of the marked cells (NeuN+/GABA+
GABAergic neurons) in each SDH and the immunoreac-
tive intensities of the staining marker (GABA), without
the description of the spinal cord laminae.
RESULTS: This method showed a significant (p<0.05)
loss of GABAergic neurons in the deeper regions on
the injured side of the SDH. This was associated with
the decrease of GABA immunoreactivity in the chronic
phase of CNPSCI (8 weeks after spinal cord injury).
CONCLUSION: This method could normalize the mor-
phometric variations and the deformations in the SDHs
in pairs in each transverse-section sample. It allowed
us to quantitatively compare the number and the dis-
tribution of marked cells in the SDHs and the average
immunoreactivities in this ellipse ROI. (Anal Quant
Cytopathol Histpathol 2021;43:8–18)
Keywords: cell count; disease models, animal;
GABA; GABA agonists; green fluorescent proteins;
immunochemistry images; immunohistochemistry;
interneurons; morphometric analysis; nerve root
compression; neuropathic pain; pain/physiopa-
Quantitative Morphometric Analysis of
Immunochemistry Images of the Spinal
Dorsal Horn
A New Technique for an Evaluation of the Spinal Dorsal
Horn in a Hemisection Spinal Cord Injury Model
Matthias Morgalla, M.D., Ph.D., Jun Li, M.D., Tianci Cheng, M.S.,
Samuel Dobrowolski, M.D., and Guilherme Lepski, M.D., Ph.D.
From the Department of Neurosurgery, University of Tuebingen, Tuebingen, Germany; and Luzhou Jiuzheng Orthopaedic Hospital,
Luzhou, Sichuan, P.R. China.
Matthias Morgalla is Professor, Department of Neurosurgery, University of Tuebingen.
Jun Li is Research Fellow, Department of Neurosurgery, University of Tuebingen, and Luzhou Jiuzheng Orthopaedic Hospital.
Tianci Cheng is Research Fellow, Department of Neurosurgery, University of Tuebingen.
Samuel Dobrowolski is Research Fellow, Department of Neurosurgery, University of Tuebingen.
Guilherme Lepski is Privatdozent, Department of Neurosurgery, University of Tuebingen.
Matthias Morgalla and Jun Li contributed equally.
This study was supported by funds of the Department of Neurosurgery, University of Tuebingen.
Address correspondence to: Matthias H. Morgalla, M.D., Ph.D., Neurosurgical Pain Clinic, Department of Neurosurgery, University of
Tuebingen, Hoppe-Seyler-Str. 3, 72076 Tuebingen, Germany (matthias.morgalla@med.uni-tuebingen.de)
(www.neurochirurgische-schmerztherapie-tuebingen.de).
Financial Disclosure: The authors have no connection to any companies or products mentioned in this article.

2. Volume 43, Number 1/February 2021 9
Analysis of Images of the Spinal Dorsal Horn
thology; pain measurement; posterior horn cells;
signal transduction; spinal cord; spinal cord inju-
ries; spinal dorsal horn.
One of the key structures which causes the signal
transmission of somatosensory and painful stim-
uli is the spinal dorsal horn (SDH).1,2 The most
favored “region of interest” (ROI) of the SDH in
neuropathic pain research extends from the lamina
I to IV. After a spinal cord injury (SCI), the phys-
iological anatomic structures of the spared spinal
cord tissues could be affected through the forma-
tion of post-SCI cysts or glial scars. These changes
in their shape can cause problems in the process
of collecting solid and comparable data when
using immunostaining images. Furthermore, non-
normalized values of the immunoreactive inten-
sity, which usually could be measured in different
images, cannot be accurately compared. Therefore,
the measurement of the immunoreactivity inten-
sities taken from the images have often been re-
placed by other quantitative analytical techniques,
such as the western blot3,4 and the real-time poly-
merase chain reaction (PCR).5,6 However, it re-
mains difficult to collect small tissue samples from
a specific area of the SDH in order to perform
quantitative tests, for instance the dissection of the
superficial laminae of SDH for the western blot
tests. Although a defined test region can be se-
lected for the quantitative measurements, e.g. real-
time PCR,7 using the technique of laser capture
microdissection (LCM),8-10 a reliable and repro-
ducible method in order to define the ROI is still
required.11
We replaced the system of the SDH laminae
with its schematic dash-line outlines by introduc-
ing an ellipse-baseline method used for the su-
perficial region of each SDH. We used fluorescent
immunohistochemistry in order to improve the
quantitative data acquisition and the analysis. By
using this method, it was possible to investigate
the SDH quantitatively and to compare the distri-
bution of immunoreactive cells and the immuno-
reactivities of the target markers in the ellipse ROI
between the injured side and the non-injured side.
Materials and Methods
The following experiments were performed:
1. Establishment of an animal model for the gen-
eration of neuropathic pain.
2. Introduction of a new technique for the quan-
titative morphometric examination of fluo-
rescent immunohistochemistry images of the
SDH.
3. Evaluation of the distribution of GABAergic
neurons and the immunoreactivities of GABA
in the SDH after SCI.
Animal Model of Neuropathic Pain
Eight male Sprague-Dawley rats (n=8, with a body
weight of 240–260 g) underwent a lateral, dorsal
hemisection at T13 using a 1 mm depth of cut. A
laminectomy was performed between the T12-13
vertebral segments. The dorsal spinal cord was
then hemisected with a No. 11 scalpel at the level
T13, just cranial to the dorsal root entry zone (Fig-
ure 1). All animal experiments were performed ac-
cording to the standards of the National Institute
of Health, the European Union Animal Care, and
the local animal care guidelines of the University of
Tuebingen (approval number: N 13/12).
Postoperative Assessment Following SCI
We used the Basso Beattie and Bresnahan (BBB)
locomotor rating scale to confirm the preservation
of the locomotor function postoperatively.12 By
using an electronic plantar Von Frey instrument
(Dynamic Plantar Aesthesiometer, Cat. No. 37400-
001, Ugo Basile, Italy), the mechanical thresholds
of the plantar surface of the rats were examined
before and after the SCI.
Figure 1 The lateral, dorsal hemisection model in the rat.

3. 10 Analytical and Quantitative Cytopathology and Histopathology®
Morgalla et al
A plantar test instrument with an infrared heat-
ing source was used for the measurement of the
thermal pain threshold of the hind paw (Plantar
Test [Hargreaves Apparatus], Cat. No. 37370-001,
Ugo Basile, Italy).
Immunohistochemistry
To investigate the pathological and cellular changes
of CNPSCI on both spinal dorsal horns, the rats were
sacrificed 8 weeks post-surgery. The 1-cm long
segments caudal to the injured side were collected
to evaluate the pain-related changes in that area.
The samples were embedded in Tissue-Tek O.C.T.
Compound (Sakura), serially and transversely sec-
tioned in a thickness of 10 µm using a cryostat
(Leica CM1520).
Following this, the slides were divided into 3
groups: below injury segment 1, 2, and 3 with 80
slides each. Double immunofluorescent labeling
with different cell markers (NeuN+GABA) was
then performed.
Quantitative Evaluations of the Superficial
SDH-Laminae with Fluorescent Images
A specifically designed ellipse ROI evaluation
method for immunofluorescent images was devel-
oped to obtain comparative and quantitative re-
sults of the distribution depth of GABAergic neu-
rons and the expression of GABA from the SDH.
Our image evaluation method is not only suitable
for the evaluations of SDH with normal physiolog-
ical architecture, but also applicable for the evalu-
ations of SDH with a structural deformation. The
entire method of image evaluation was performed
using the software ImageJ (ImageJ 1.47v, National
Institutes of Health, Bethesda, Maryland, USA).
Setting Up a Standard Ellipse ROI with Three
Baselines for Evaluating Target Markers
To apply this method, the markers of NeuN and
GABA were used. Originally, two images with a
neuronal marker—NeuN—and another marker—
GABA—from one double-labeled SDH sample
were loaded together onto the ImageJ. The NeuN
image was fused with the GABA image with a
100% opacity. Now the fused image was ready for
setting up the ellipse ROI with its three baselines,
at both the injury side and the non-injury side.
• At first, the most superficial NeuN-positive
neurons at the distal outline of SDH (Figure
2A, arrowheads) were identified. An ellipse
ROI was drawn to keep these neurons at-
tached to the outline of the ellipse region. The
NeuN-positive neurons in the lateral spinotha-
lamic tract, which is close to the superficial
SDH, should be carefully excluded (Figure 2A,
yellow arrow). This was vital. However, the
lateral spinothalamic tract could be excluded
safely in all our cases.
• Secondly, the baseline one (BL1) (Figure 2A,
yellow dashed line) was established along the
axis of the ellipse ROI. Two more baselines
were then introduced in addition to the BL1:
baseline 2 (BL2) (Figure 2A, red dashed line),
which is a tangent line at the distal end of the
ellipse parallel to BL1.
• The third baseline was established (BL3) (Fig-
ure 2A, blue dashed line), which is the verti-
cal line between BL1 and BL2. This ellipse ROI
could now be loaded onto the image of GABA
by removing the previously fused NeuN image
(Figure 2B).
Calculation of the Ellipse Area as the Reference
Factor in Order to Compensate for the
Morphometric Deformation
A size reference factor (SRF) for each SDH was
calculated by computing the ellipse area with half
the size of BL1 and the BL3. The SRF on the non-
injured side was used as the standard size refer-
ence factor for both sides on the sample slice.
We used the following formula in order to cal­
culate the SRF:
Size Reference Factor (SRF)=Ellipse areas=π×1/2
BL1×BL3.
In order to eliminate the errors which were
caused by the lesion-induced structural deforma-
tion, the original value of the injured side was al-
ways normalized by this slice-unique SRF before
performing the further measurements or the sta-
tistical analysis (Figure 2C).
The formulas to calculate the normalized cell
number and cell depth of GABAergic neurons are
Normalized cell number.injured side=Total cell number.
injured side÷SRF
Normalized cell number.non-injured side=Total cell num-
ber.non-injured side÷SRF
Normalized cell depth.injured side=Total cell depth.injured
side÷SRF
Normalized cell depth.non-injured side=Total cell depth.non-
injured side÷SRF.

4. Volume 43, Number 1/February 2021 11
Analysis of Images of the Spinal Dorsal Horn
Image Processing for Evaluating the Distribution of
GABAergic Neurons by Using the Ellipse-Baseline
Method
In this study, the location and the number of
GABAergic neurons in the superficial laminae of
the SDH were evaluated by using the ellipse-
baseline method. In order to achieve an optimal
identification of GABAergic neurons in the SDH,
firstly the fluorescent signals of both GABA (Fig-
ure 3A) and NeuN (Figure 3B) were converted to
Figure 2
The setup of a standard ellipse
ROI with three baselines.
(A) Setup of the ellipse ROI
with the image of neuronal
marker (NeuN). (B) Removal
of the overlapped image of a
neuronal marker, and loading
of the ellipse ROI onto the
image of the interesting
marker, e.g. GABA.
BL = baseline, arrow heads =
the most superficial NeuN-
positive cells, Arrow = the
NeuN-positive cells in the
lateral spinal nucleus. Scale
bar = 100 μm. (C) The
overview of the ellipse ROIs
in the spinal dorsal horns with
the neuronal marker (NeuN).
The ellipses from the two
sides are different due to the
deformation on the injury side
following the spinal cord
injury. The size of the ellipse
on the non-injury side was
used for normalization of the
data from both sides. Scale
bar = 100 μm.

5. 12 Analytical and Quantitative Cytopathology and Histopathology®
Morgalla et al
clear and separated single-cell signals by using a
plugin, Fast Filters (Filter Type: maximum; Pre­
processing: none; Subtract Filtered), in ImageJ,
respectively. Then, these two images with the
converted color were merged, followed by setting
up a standard ellipse ROI with three baselines
as described above (Figure 3C). Thereafter, the
double-stained cells within the ellipse ROI were
marked as GABAergic neurons on the merged
picture (Figure 3C). Immediately after marking all
the GABAergic neurons, the number of marked
cells was recorded. The vertical distance from
each marked cell to BL2 was measured as well
(Figure 4). The vertical distance describes the dis-
tance from the surface of the ellipse to its center.
It serves as a measure of the depth of the cells
within the spinal dorsal horn. It is therefore an
important measurement in order to locate the dif-
ferent cells.
The loss of the deeply located GABAergic inter-
neurons on the injured side in comparison to the
non-injured side could be quantitatively described
in our study of the SCI-induced neuropathic pain
model through the measurement of the vertical dis-
tances from each target cell to the BL2.
Evaluating the Fluorescent Intensities of the Target
Marker in the Ellipse ROI
In the current study, the fluorescent intensity of
GABA was measured in the ellipse ROI of the
SDH. Firstly, three separate layers (NeuN, GABA,
and DAPI, respectively) of the same image were
Figure 3 Locating the GABAergic neurons in the ellipse ROI by using signal-filtered images. (A) The filtered signal of GABA
immunoreactivity. The red and purple arrows indicate strong signals of GABA immunoreactivity. (Aa) The magnified area from picture A.
(B) The filtered signal of NeuN immunoreactivity. The red arrow indicates the lack of NeuN immunoreactivity, and the purple arrow
indicates an obvious NeuN immunoreactivity. (Bb) The magnified area from picture B. (C) Locating the GABA-immunoreactive neurons
with numeric blue spots of the Cell Counter. The red arrow indicates the exclusion of a simultaneous GABA-positive and NeuN-negative
signal, and the purple arrow indicates a GABA-positive and NeuN-positive neuron. (Cc) The magnified area from picture C. Scale bar =
100 μm.

6. Volume 43, Number 1/February 2021 13
Analysis of Images of the Spinal Dorsal Horn
loaded together to ImageJ. The ellipse ROI was
drawn with the neuron marker NeuN on the
SDH as described above (Figure 5A), and then the
layer of NeuN (Figure 5B) was removed. There-
after, two important zones of background sig-
nals were confirmed. Background one (BG 1) (Fig-
ure 5B, square zone), which was a blank area
with no involvement of any tissue fragments, was
created for generating a basic non–tissue-related
fluorescent intensity. Background two (BG 2) (Fig-
ure 5B, long strip area, located on the layer of
DAPI), which was the transection zone of the
nerve root close to the SDH, was applied to gen-
erate a tissue-related background data of fluores­
cent intensity. Then, the DAPI layer would be re-
moved, and the ellipse ROI and the background
zones were left on the layer of GABA. The mean
value of the fluorescent intensity of GABA in
these three zones was measured in ImageJ (Fig-
ure 5B).
Based on the the following calculation, the final
fluorescent intensity of each ROI was generated
for the following normalization:
DFI=MFIROI ÷MFIBG1 ÷MFIBG2,
where DFI=data of fluorescent intensity, MFI=mean
fluorescent intensity, ROI=the region of interest,
BG1=background one, and BG2=background two.
The normalization for compensating the errors
Figure 4 Measuring the location depth of GABAergic neurons.
BL = baseline. Scale bar = 100 μm.
Figure 5 Setting up a standard ellipse ROI with two regions of background for evaluating the immunoreactivity of specific markers.
(A) Setting up the ellipse ROI as described above. (B) Setting up the region of background 1 and 2 in the blank area and transection zone
of a nerve root, respectively. Red = NeuN, Green = GABA, Blue = DAPI. BG = background, ROI = the region of interest, NR = nerve root.
Scale bar = 100 μm.

7. 14 Analytical and Quantitative Cytopathology and Histopathology®
Morgalla et al
which were induced by the structural deformation
was performed by using the following formulas
with SRF:
Normalized DFIinjured side =DFIinjured side ÷SRF
Normalized DFInon-injured side =DFI non-injured side ÷SRF,
where DFI=data of fluorescent intensity and
SRF=size reference factor.
Thereafter, the normalized data of fluorescent
intensities from the left and the right SDH were
compared.
Statistics
All the values in this study were described as
mean±SD. All the statistical analyses were per-
formed by using SPSS 13.0 for Windows. Before
doing the statistical analyses, all the original values
were normalized as we described above.
The difference of the depth distribution of
GABAergic neurons in the ellipse ROI between
the injured side and the non-injured side was in-
vestigated by the two-independent-samples tests
of the nonparametric test followed by the Mann-
Whitney test. To compare the differences of the
number of GABAergic neurons, fluorescent in-
tensities of GABA-only pairwise values from the
injury side and non-injury side of each spinal
cord sample were analyzed with the two-related-
samples tests of the nonparametric test followed
by Wilcoxon signed-rank tests. The level of signifi-
cance was p<0.05.
Results
The motor function of both hind limbs (injury
side and control side) was evaluated weekly from
the first day after the spinal cord injury. After
initial impairment of both hind limbs, the limb
of the injured side recovered after two weeks to
15.55±1.45 and after 5 weeks to 19.22±1.18 (Fig-
ure 6).
The post-SCI mechanical pain threshold base-
line value as a marker for mechanical allodynia
was measured one day before surgery and then
assessed weekly for eight weeks. A substantial
decrease of the mechanical pain threshold of the
hind limb on the injury side was noted during the
first week post-SCI. We found a drop of 40.96%±
7.32 when we compared this result to the baseline
Figure 6
The changes of the Basso
Beattie and Bresnahan (BBB)
score of both hind limbs. The
horizontal axis indicates the
weeks after spinal cord injury
(SCI), starting at the time of
surgery. The dashed line of
the BBB scale 21 indicates
the complete recovery of
motor function; the dashed
line of BBB scale 13 indicates
the ability to consistently
stride with weight bearing;
the dashed line of the BBB
scale 7 indicates the extensive
movements of all three joints
of the rear limb. *p<0.05,
mean±SD.

8. Volume 43, Number 1/February 2021 15
Analysis of Images of the Spinal Dorsal Horn
value. This was taken as evidence for the existence
of mechanical allodynia (Figure 7).
The thermal pain threshold dropped by 29.71%±
2.63% during the first post-SCI week on the injury
side, which indicates the appearance of thermal
allodynia (Figure 8).
Distribution of GABAergic Neurons in the SDH
The depth distribution of GABAergic neurons in
the SDH was evaluated by measuring the verti-
cal distance between a specific GABAergic neu-
ron (GABA+ and NeuN+) and BL2. The nor-
malized numbers of GABAergic neurons in each
SDH were analyzed as well. From each below-
injury segment of the 8 rats, respectively, the
depth of each GABAergic neuron and the num-
ber of GABA-positive neurons were both record-
ed. We investigated the depth distribution by
measuring the vertical depth of each GABAergic
neuron at the injured side and the non-injured
side and grouped the results. The normalized
value of the depth distribution at the injured
and the non-injured side was 0.96±0.09 mm/
mm2 and 1.32±0.18 mm/mm2, respectively. No-
tably, 28% of the GABA-expressing neurons
were lost at the deepest point on the injury side
(p<0.05).
We evaluated the number of GABAergic neu-
rons and counted and grouped the number of
GABAergic neurons in each SDH on the injury
side and the non-injury side. The values from both
sides were normalized and compared in pairs in the
same sample.
The normalized number of cells of GABAergic
neurons on the injured and the non-injured side
was 193.54±31.28/mm2 and 321.12±11.53/mm2,
respectively. The difference between the two sides
was significant (p<0.05) (Figure 9).
The normalized values of the intensity of the
GABA expression in the ellipse ROI on the in-
jured and non-injured side were 4.05±0.81/mm2
and 8.39±1.31/mm2, respectively. The GABA in-
tensity of the ellipse ROI on the injured side had
been decreased by about 52% in comparison to
that of the non-injured side (p<0.05). This result
was consistent with the decrease of the number
of GABAergic neurons in the SDH on the injured
side.
Figure 7
The changes of the thermal
pain thresholds following a
spinal cord injury (SCI). When
the thermal threshold had
decreased by more than
25% (dashed line indicates
75% of the normal thermal
threshold), a thermal
neuropathic pain was present.
*p<0.05, mean±SD.

9. 16 Analytical and Quantitative Cytopathology and Histopathology®
Morgalla et al
Discussion
In this study, we created an ellipse ROI for the
superficial region of each SDH in accordance with
the distribution of NeuN-positive neurons with-
out the description of the spinal cord laminae. A
reliable data normalization was established addi-
tionally with the aid of a series of compensatory
factors and two background zones. SDH images
could now be quantitatively and morphometrical-
ly analyzed. This includes fluorescent images but
also other SDH images with marker-combinations
which are based on at least one neuronal marker.
There are two important requirements to utilize
this method:
• The spinal cord injury must be restricted to one
side only.
• A neuronal marker has to be used to identify
the edge of the SDH.
The Lack of Methods Which Could Quantitatively
and Morphometrically Analyze the Images of the
SDH
There are no standardized methods available in
order to quantitatively analyze and compare the
different cell populations in the spinal cord after
trauma.13 The problem with trauma is that the
anatomy of the two sides of the cord may have
Figure 8
The changes of mechanical
pain thresholds following a
spinal cord injury (SCI). When
the mechanical threshold
decreased by more than 25%
(dashed line indicates 75%
of the normal mechanical
threshold), mechanical
neuropathic pain was present.
*p<0.05, mean±SD.
Figure 9 A bar graph illustrating the numbers of GABAergic
neurons per mm2 in the ellipse ROI of the injured and non-
injured side. There was a significant reduction of GABAergic
neurons on the injured side. *p<0.05, mean±SD.

10. Volume 43, Number 1/February 2021 17
Analysis of Images of the Spinal Dorsal Horn
able to have a quantitatively comparative inves­
tigation directly from above the SDH and would
then be aware of the exact target location as well.
Another inconvenience is the establish­
ment of 2
different zones of background signals in order to
evaluate the fluorescent intensities of the target
marker in the ellipse ROI. However, these are
important in order to ensure a proper evaluation of
the fluorescent intensities.
Conclusion
The SDH plays a key role in the development
and the persistence of chronic neuropathic pain
following a spinal cord injury. This morphometric
method allowed us to directly and quantitative-
ly investigate the differences of the cellular and
molecular changes in the superficial spinal laminae
between the injured and non-injured spinal dorsal
horn. This method showed a significant (p<0.05)
loss of GABAergic neurons in the deeper regions
on the injured side of the SDH. This was associ-
ated with the decrease of GABA immunoreactiv-
ity in the chronic phase of CNPSCI (8 weeks after
spinal cord injury). This ellipse-baseline method of
in situ morphometric analysis will also be useful
to other scientists for the provision of more details
with regard to the interaction between the neurons
and the glial cells.
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changed, and because of this they are difficult to
compare with one another.
Many studies show photographs of the spinal
cord which were then divided into segments such
as laminae I–II, II–IV, V–VI, and the ventral horn.14
This is also a possible approach as long as the
two sides of the spinal cord remain unaltered.
However, in many cases it seems difficult to iden-
tify the single laminae and to define areas which
can be compared with one another. In another
study which examines the medullary dorsal horn,
a rectangular cell counting window was used for
the evaluation of the cells. This was similar to our
approach, and this allowed comparable cell counts
in different slices.15 A method used to count cells
quantitatively is the disector method, which is now
known as the “physical disector.”16 It identifies
profiles of a particle seen in one section but not
in another (tops). The advantage of the disec-
tor method is that an unbiased estimate can be
obtained. The physical disector method was used
in many studies to count the neurons in the su-
perficial laminae of the dorsal horn of rodents.17-20
In another study the physical disector analysis
was used in a nerve constriction model. Photo-
graphs were taken of different sections and mon-
tages were reconstructed. The midpoints of the
lamina II–III and the III–IV boundaries were con-
nected with a straight line in order to improve the
cell count.21
In a contusion model of the cervical spine of
mice all of the immunostainings were quantified in
the most superficial laminae (I–II) or the lamina III
of the spinal cord in the dorsal horn.22 However,
a size reference factor for the comparison was not
calculated.
Therefore, with the traditional boundary draw-
ing methods a morphometric comparison between
injured and non-injured SDHs is difficult or often
not possible and an improvement is still needed.
Disadvantages and Inconveniences of This Method
In this method the SDH was considered in its
entirety to investigate the distributions and active
intensities of all the cell types. However, the
cells in the SDH are located in different laminae,
with different functions. The results generated
from our current method can only make a com-
parison possible on a full scale. In future studies
we would like to introduce the SDH laminae
with accurately defined outlines into this ellipse-
baseline method. The researchers would then be

11. 18 Analytical and Quantitative Cytopathology and Histopathology®
Morgalla et al
J Comp Neurol 2014;522(2):393-413
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