STUDY PROTOCOL
1.0 TEST SYSTEM DETAILS:
Specie: Mus musculus (Mouse)
Strain : C57BL/6; Balb/c; Swiss albino
Age : 6-10 weeks
Body Wight : 25-30 g
Sex : Male/Female
No. of animals : 10 /Group
Total Animals : 37+3 Extra= 40
Specie: Mus musculus (Mouse)
Strain : C57BL/6; Balb/c; Swiss albino
Age : 6-10 weeks
Body Wight : 25-30 g
Sex : Male/Female
No. of animals : 10 /Group
Total Animals : 37+3 Extra= 40
2.0 ALLOCATION OF GROUPS:
*The doses and ROA (Routes of administration) will be decided based on the type of reference drug
# 3 extra animals will be taken extra due to mortalities during the surgery
3.0 METHOD:
· Mice will be anesthetized with Isoflurane.
· Mice will be placed on a water-re-circulating heating pad under a surgical microscope
· 5 microliters of tropicamide (dilation) & 5 microliters of proparacaine (topical anesthetic) will be topically administered to each eye using a micropipette.
· Any excessively long eyelashes will be trimmed with vans scissors.
· The central cornea will be incision injury and followed by 2 sutures with 11-0 nylon.
· Topical bacitracin antibiotic ointment will be applied to the eye surface.
· Mouse returned to the cage and monitored until able to ambulate.
· Mice will be checked the following days and an antibiotic ointment will be re-applied.
· To determine the effect of the plant extract (Formulation) four treatment groups will be studied: a plant extract group (Formulations), a vehicle control group, a positive control group (Avastin), and a group that did not receive suture placement (“normal cornea” group). The corresponding treatments were applied as Eye drop on the cornea on days from 0 through 14 days
· Microscopic examination of neo-vascularization and fibrosis induction will be done every day using stereomicroscope observation.
· Corneas will be harvested on day 15. Whole mounts of neovascularized corneas will be stained for histology.
· Fluorescent microscopy will be used to digitally quantify the area and volume of neovascularization and fibrosis.
4.0 ENDPOINT PARAMETER(S):
· Clinical microscopic and macroscopic observation and images were taken every day in all study groups to assess possible gross corneal angiogenesis and fibrosis.
· Histopathology and immunohistochemistry of cornea tissue.
5.0 REFERENCE(S):
· Maddula S Davis DK Maddula S Burrow MK Ambati BK . Horizons in therapy for corneal angiogenesis. Ophthalmology. 2011;118:591–599.
· Rocher N Behar-Cohen F Pournaras JA . Effects of rat anti-VEGF antibody in a rat model of corneal graft rejection by topical and subconjunctival routes. Mol Vis. 2011;17:104–112
· Cursiefen C Chen L Borges LP . VEGF-A stimulates lymphangiogenesis and hemangiogenesis in inflammatory neovascularization via macrophage recruitment. J Clin Invest. 2004;113:1040–1050
· Santos LN de Moura LR Fernandes BF Cheema DP Burnier MNJr Histopathological study of delayed regraft after corneal graft failure. Cornea. 2011;30:167–170.
· Weisbrod DJ Sit M Naor J Slomovic AR . Outcomes of repeat penetrating keratoplasty and risk factors for graft failure. Cornea. 2003;22:429–434.
· Olsson AK Dimberg A Kreuger J Claesson-Welsh L . VEGF receptor signaling: in control of vascular function. Nat Rev Mol Cell Biol. 2006;7:359–371.
· Ferrara N Kerbel RS . Angiogenesis as a therapeutic target. Nature. 2005;438:967–97.
· Wu FTH Stefanini MO Mac Gabhann F Kontos CD Annex BH Popel AS . A systems biology perspective on sVEGFR1: its biological function, pathogenic role and therapeutic use. J Cell Mol Med. 2010;14(3):528–552; doi:
· Shibuya M . Structure and dual function of vascular endothelial growth factor receptor-1 (Flt-1). Int J Biochem Cell Biol. 2001;33:409–420.
· Shibuya M . Differential roles of vascular endothelial growth factor receptor-1 and receptor-2 in angiogenesis. J Biochem Mol Biol. 2006;39:469–478
· Kerber M Reiss Y Wickersheim A . Flt-1 signaling in macrophages promotes glioma growth in vivo. Cancer Res. 2008;68:7342–7351.
· Huckle WR Roche RI . Post-transcriptional control of expression of sFlt-1, an endogenous inhibitor of vascular endothelial growth factor. J Cell Biochem. 2004;93:120–132. Rahimi N Golde TE Meyer RD . Identification of ligand-induced proteolytic cleavage and ectodomain shedding of VEGFR-1/FLT1 in leukemic cancer cells. Cancer Res. 2009;69:2607–2614.
|
Groups |
Treatment |
Dose;
ROA |
No.
of Animals |
|
G1 |
Normal Control (No suture) |
Normal saline or
0.25% Na-CMC |
8 |
|
G2 |
Suture Control |
Normal saline or
0.25% Na-CMC |
8 |
|
G3 |
Reference Drug-
Avastin |
10µg/mouse (10µl) |
8 |
|
G4 |
Plant
Extract/Herbal Formulation-1 |
X mpk; |
8 |
|
G5 |
Plant
Extract/Herbal Formulation -1 |
XX mpk; |
8 |
*The doses and ROA (Routes of administration) will be decided based on the type of reference drug
# 3 extra animals will be taken extra due to mortalities during the surgery
3.0 METHOD:
· Mice will be anesthetized with Isoflurane.
· Mice will be placed on a water-re-circulating heating pad under a surgical microscope
· 5 microliters of tropicamide (dilation) & 5 microliters of proparacaine (topical anesthetic) will be topically administered to each eye using a micropipette.
· Any excessively long eyelashes will be trimmed with vans scissors.
· The central cornea will be incision injury and followed by 2 sutures with 11-0 nylon.
· Topical bacitracin antibiotic ointment will be applied to the eye surface.
· Mouse returned to the cage and monitored until able to ambulate.
· Mice will be checked the following days and an antibiotic ointment will be re-applied.
· To determine the effect of the plant extract (Formulation) four treatment groups will be studied: a plant extract group (Formulations), a vehicle control group, a positive control group (Avastin), and a group that did not receive suture placement (“normal cornea” group). The corresponding treatments were applied as Eye drop on the cornea on days from 0 through 14 days
· Microscopic examination of neo-vascularization and fibrosis induction will be done every day using stereomicroscope observation.
· Corneas will be harvested on day 15. Whole mounts of neovascularized corneas will be stained for histology.
· Fluorescent microscopy will be used to digitally quantify the area and volume of neovascularization and fibrosis.
4.0 ENDPOINT PARAMETER(S):
· Clinical microscopic and macroscopic observation and images were taken every day in all study groups to assess possible gross corneal angiogenesis and fibrosis.
· Histopathology and immunohistochemistry of cornea tissue.
5.0 REFERENCE(S):
· Maddula S Davis DK Maddula S Burrow MK Ambati BK . Horizons in therapy for corneal angiogenesis. Ophthalmology. 2011;118:591–599.
· Rocher N Behar-Cohen F Pournaras JA . Effects of rat anti-VEGF antibody in a rat model of corneal graft rejection by topical and subconjunctival routes. Mol Vis. 2011;17:104–112
· Cursiefen C Chen L Borges LP . VEGF-A stimulates lymphangiogenesis and hemangiogenesis in inflammatory neovascularization via macrophage recruitment. J Clin Invest. 2004;113:1040–1050
· Santos LN de Moura LR Fernandes BF Cheema DP Burnier MNJr Histopathological study of delayed regraft after corneal graft failure. Cornea. 2011;30:167–170.
· Weisbrod DJ Sit M Naor J Slomovic AR . Outcomes of repeat penetrating keratoplasty and risk factors for graft failure. Cornea. 2003;22:429–434.
· Olsson AK Dimberg A Kreuger J Claesson-Welsh L . VEGF receptor signaling: in control of vascular function. Nat Rev Mol Cell Biol. 2006;7:359–371.
· Ferrara N Kerbel RS . Angiogenesis as a therapeutic target. Nature. 2005;438:967–97.
· Wu FTH Stefanini MO Mac Gabhann F Kontos CD Annex BH Popel AS . A systems biology perspective on sVEGFR1: its biological function, pathogenic role and therapeutic use. J Cell Mol Med. 2010;14(3):528–552; doi:
· Shibuya M . Structure and dual function of vascular endothelial growth factor receptor-1 (Flt-1). Int J Biochem Cell Biol. 2001;33:409–420.
· Shibuya M . Differential roles of vascular endothelial growth factor receptor-1 and receptor-2 in angiogenesis. J Biochem Mol Biol. 2006;39:469–478
· Kerber M Reiss Y Wickersheim A . Flt-1 signaling in macrophages promotes glioma growth in vivo. Cancer Res. 2008;68:7342–7351.
· Huckle WR Roche RI . Post-transcriptional control of expression of sFlt-1, an endogenous inhibitor of vascular endothelial growth factor. J Cell Biochem. 2004;93:120–132. Rahimi N Golde TE Meyer RD . Identification of ligand-induced proteolytic cleavage and ectodomain shedding of VEGFR-1/FLT1 in leukemic cancer cells. Cancer Res. 2009;69:2607–2614.
END OF THE DOCUMENT
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