animal study

1. TOPICAL SPRAY FOR STANDARDIZED
SENNAALATA LEAF EXTRACT
Student Name: Vijaykumar Meti
Student ID NO: QIUP-2017-10-001457
Post Graduate Level: PhD (Pharmaceutical Sciences)
9/13/2022
1
ANIMAL STUDY FOR SENNAALATA LEAF EXTRACT

2. IN-VIVO MODEL FOR FUNGAL INFECTION
IN SKIN
 Animals are divided into 5 groups (8 animals in
each group):
1. Group-I: -Ve control (Healthy animals).
2. Group-II: +ve Controll Treated by Standard Drug
(Ketocanazole).
3. Group-III: Test Group Treated by formulation
containing Senna alata L extract.
4. Group-IV: Treated with Senna alata L. Extract
5. Group V: Treated with Placebo Formulation

3. Materials for In vivo Model Studies
 Animal- 5-7 week old C57BL/6 mice.
 Fungal strain- Candida albicans pseudohyp.hae, CAF2-1 strain.
 YPD medium containing 10%(v/v) Fetal bovine serum (FBS)
 10% neutral buffered saline.
 Liquid nitrogen.
 Phosphate buffered saline, sterile.
 Inverted microscope.
 Electric razor.
 Hemacytometer.
 1-tuberculin syringe and 25-G needle.
 Dissectng pins.
 Sterile dissecting tools including straight scissors and forceps
4mm punch biopsy tool
 Pellet pestles

4. Dermal/cutaneous candidiasis in Adult Mice
 Preparing mice for infection:
1. Mice should be shaved at dorsal region at least 24h before injection.
2. Mice should be individually caged while inoculating so as to designate mice
for longitudinal clinical study or histology and fungal burden assays.
3. Candida albicans culture is prepared by keeping overnight and adjusted the
culture for inoculating at density of 5 x 104 cells/ml and 5x106 cells/ml in
YPD with 10% FBS.
4. Incubate the culture for 37°C for 2h, and should confirm the growth of
pseudohyphae by microscopic examination using a hemacytometer, so as to
confirm that at least 95% of blastospores have turned to the virulent
pseudohyphae form
5. Pseudohyphae later is injected in a 50µl volume of phosphate buffer saline
intradermally into the deep dermis and superficial fat of the shaved dorsal
region of each mouse using 25-G needles. Inject a separate group with PBS
for Control.
6. Proper injection will result in the formation of a red or swollen mark on the
skin of mice.
7. Attempts should be made to place the injection at the same location for each
mouse to aid in determination of healing and harvesting tissue at the relevant
tissue site

5. Diagnostics of candida albicans infected mice
 Check mice visually and with photographs at
least three times weekly at injection site to see
any kind of nodules, ulcerations, erythema and
crusting.
 Score disease positively if any of the symptoms is
present.

6. READ OUTS FOR DISEASE
1. Healing rates.
2. Fungal burden.
3. Leukocyte infiltration.
4. Epidermal hyperplasia.

7. Protocol For Healing Rate of Fungal Infected Mice
 Incision model of wound healing: On skin wound
healing process will be analyzed by tracing the
wound closure rate on day 2, 4, 6, 8, 10, 12, 14 and
16 using transparent paper and permanent marker.

8. Determination of fungal burden assay
 Determination of fungal burden assay is a histological
study. Which is studied based on molecular mechanism
using RT-PCR or microbial activity by counting Colony
forming unit(CFU) in a plate.
 Determination of fungal tissue burden is straightforward for
yeast cells and a sensitive measure for the progression of
infectious process.
 Quantitation of fungal hyphae is more complicated and a
universal standard does not exist.
 Colony forming unit (CFU) determinations do not scale
linearly scale with hyphal burden in infected tissues.
 Quantitative measurements of fungal DNA (using the 18S
rRNA gene as a target) have been developed using
polymerase chain reaction-based methods on DNA
extracted from homogenized lung tissue.
 The release of fungal antigens during vertebrate tissue
invasion by enzyme-linked immuno-absorbent assays also
provides a quantitative readout for fungal tissue burden in

9. Protocol for fungal burden determination of
fungal induced mice
 For fungal burden determination, 5x106 cells/ml should be inoculated,
while an inoculum of 5 x 104 cells/ml is used to ensure recovery of
intact skin tissue for histology.
 For fungal burden determination, sacrifice mice on Day 4.
 pin the euthanized mouse to a clean surface with shaved dorsal region
facing up
 Elevate skin with sterilized forceps and snip the skin, place scissors in
the incision, and separate skin from the deeper layers by blunt
dissection.
 Cut through skin and remove entire area of shaved skin.
 Place tissue, epdermis side down, on firm sterile surface and excise a
4mm punch section of skin where injection site was located.

10. Continued..
 For histology, place tissue in 1ml of 10% neutral buffered formalin, for
RT- PCR, place tissue in 1ml RNA later on ice or flash frozen in liquid
nitrogen.
 Weigh tissue section being used for fugal burden assay and place in
200µl of PBS and homogenize with a pellet pestle on ice.
 Plate 100µl of tissue samples dilutions on 9cm sabourauds dextrose
agar plates containing 50µg/ml chloramphenicol. Incubate plates upside
down at 37°C for 48h.
 Count the number of colonies and calculate as colony forming units/g
tissue.

11. Leukocyte infiltration of fungal induced mice
 It is the investigation of molecular and cellular
mechanism which includes the pathologies of skin.
 Inexpensive, efficient, and simple protocols for the
enzymatic digestion of mouse skin tissue can provide
preparations of cells that can be used for a variety of
downstream applications to better understand the
pathophysiology of skin diseases.
 It is a simple and economical method describes
enzymatic digest of mouse skin tissue and isolation of
skin infiltrating leukocytes that can be used for cell
culture, in vivo adoptive transfer, flow cytometric
analysis and sorting or gene expression studies.

12. Procedure to prepare a single cell suspension of
skin-infiltrating leukocytes with high cell viability
 Existing skin tissue dissociation methods may result in
low cell viability and surface marker integrity, or require
custom enzyme kits and expensive tissue dissociation
machines.
 The digestion of mouse ear skin tissue is reasonably
prevalent, digesting highly keratinized skin tissue (e.g.
from the flank) can result in cell preparations
contaminated with large amounts of non-cellular debris.
 An ideal protocol for tissue dissociation should aim for
high cell viability, low levels of cell aggregation, and
minimal damage to cell surface proteins. High quality
lymph node stromal cell preparations should be
accomplished with protocols that use shorter enzyme
incubations, Ca2+ and Mg2+ free media, and avoid trypsin
and dispase

13. Continued…
 protocol is described to dissociate, isolate, and enrich skin-
infiltrating leukocytes from allergen-challenged mouse flank
skin.
 Excised skin is pre-incubated in Hank's Balanced Salt Solution
(HBSS) with 10% fetal bovine serum for 1 hr to soften the tissue
for digestion and remove any excess dead skin or fatty tissue.
 Followed by a 30 min enzymatic digestion step with 0.7 mg/mL
collagenase D. Collagenase D has minimal effects on density of
cell surface markers, and no effect on T cell proliferation In
vitro16,18, making it highly suitable for applications involving the
characterization of surface proteins.
 Enzymatic digestion, discontinuous density gradient
centrifugation was used to remove epithelial cells and debris
from the single-cell suspension and enrich for hematopoietic
cells.
 protocol used to isolate leukocytes from flank skin challenged
three times with the hapten oxazolone (Ox) in previously
sensitized mice .
 Cells were analyzed using multi-parametric flow cytometry to

14. Critical parameters of fungal induced mice
 The standard dermal model suggests monitoring mice
three times a week for healing. Depending on the
knockout strain of mouse being tested, daily
monitoring may be necessary. Fungal load is quite
variable in this model, so fairly large group may be
needed to observe statistically significant differences.

15. Expected results for fungal induced mice
 At the site of injection, a wound forms within 24h to
48h of inoculation with candida albicans.
 Typical lesions will first present as a nodule with
ulceration and redness, with crusting occuring as the
lesion heals.
 Wild type mice inoculated with candida albicans
dermally clear the organism completely by Day 20,
with complete wound healing by this point.

16. Time considerations for fungal induced mice
 Mice need to be shaved 24h before infection.
 Infection preparation is the same as in the other
models, except extra time must be allotted to allow
incubation of pseudohyphae.
 Intradermal injections do not require anesthesia, but
speed depends on the skill of the experimenter to
inject mice that are not sedated.
 In dermal model, mice must be monitored and
measure for healing for an extended period (minimum
to weeks)