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Potential role of intravitreal human placental stem cell implants in inhibiting progression of diabetic retinopathy in type 2 diabetes: neuroprotective growth factors in the vitreous.

Scalinci SZ, Scorolli L, Corradetti G, Domanico D, Vingolo EM, Meduri A, Bifani M, Siravo D - Clin Ophthalmol (2011)

Bottom Line: Intravitreal injection of human mesenchymal stem cells has been shown to be effective in slowing the progression of diabetic retinopathy in an animal model of chemically induced diabetes mellitus.Twenty-two Lewis rats were treated with an intravitreal human mesenchymal stem cell microinjection.Increased intravitreal and retinal concentrations of neuroprotective growth factors in rats confirm the neuroprotective activity of human mesenchymal stem cells in diabetic retinopathy.

View Article: PubMed Central - PubMed

Affiliation: Glaucoma and Low Vision Study Center, Department of General Surgery and Organ Transplants, University of Bologna, Bologna. sergio.unibo@unibo.it

ABSTRACT

Background: Intravitreal injection of human mesenchymal stem cells has been shown to be effective in slowing the progression of diabetic retinopathy in an animal model of chemically induced diabetes mellitus. We studied changes in growth factor levels released from human mesenchymal stem cells in the vitreous cavity as well as changes in growth factor levels in host retinal neurons following intravitreal injection.

Methods: Twenty-two Lewis rats were treated with an intravitreal human mesenchymal stem cell microinjection. Determination of neurotrophic factors released by human mesenchymal stem cells in the vitreous was carried out using real-time polymerase chain reaction.

Results: Detectable levels of neurotrophic factors were identified postoperatively in the vitreous of all rats.

Conclusion: Increased intravitreal and retinal concentrations of neuroprotective growth factors in rats confirm the neuroprotective activity of human mesenchymal stem cells in diabetic retinopathy.

No MeSH data available.


Related in: MedlinePlus

Variation in water consumption and glycemia after treatment with streptozotocin. Rats treated with streptozotocin demonstrated a notable increase in daily water consumption, up to values of 150–200 mL/day at 21 days after treatment (mean 163.99 ± 1.9 mL/day), while reference values were 20–23 mL/day. Rats treated with streptozotocin reached mean glycemic levels of 250–350 mg/dL at 21 days after treatment (301.87 ± 4.1 mg/dL) unlike the nondiabetic rats (standard values 110–130 mg/dL).
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f1-opth-5-691: Variation in water consumption and glycemia after treatment with streptozotocin. Rats treated with streptozotocin demonstrated a notable increase in daily water consumption, up to values of 150–200 mL/day at 21 days after treatment (mean 163.99 ± 1.9 mL/day), while reference values were 20–23 mL/day. Rats treated with streptozotocin reached mean glycemic levels of 250–350 mg/dL at 21 days after treatment (301.87 ± 4.1 mg/dL) unlike the nondiabetic rats (standard values 110–130 mg/dL).

Mentions: This preliminary interdisciplinary multicenter study was supported by the University of Bologna and carried out by the Department of General Surgery and Organ Transplants at the University of Bologna in collaboration with the Department of Ophthalmology at the University of Naples, and with the support of the Department of Ophthalmology at the US Army Health Clinic Camp Darby of Livorno. This study was approved by the ethical committee at the Sant’Orsola-Malpighi Hospital, University of Bologna, and all procedures were carried out under an institutionally approved protocol in accordance with the ethical principles and standards of the Federation of European Animal Science Association, following the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the current legislation on preclinical research in Italy. All the regulations of the Centralized Veterinary Service of Bologna were respected. Fifteen young adult male syngenetic Lewis rats, weighing 180–200 g (mean 188.56 ± standard deviation [SD] 7.14 g) were included in this randomized preclinical study and were rendered diabetic using streptozotocin. Glycemia10 was set at 90–120 (mean 98.3 ± 3.4) mg/dLat baseline (time 0, beginning of the study). Animals which did not meet these requirements were excluded. All rats selected for study were further subdivided into two groups, ie, Group A and Group B. Group A (n = 12) underwent an intravitreal human placental stem cell implant, and Group B (n = 3) served as the control. The rats in both groups were rendered diabetic by a single intraperitoneal injection of streptozotocin11 65 mg/kg. Starting from the day after streptozotocin injection, evening glycemia was evaluated daily and rats with a glycemia index ≥250 mg/dL for 3 consecutive days were considered to have developed type 2 diabetes mellitus. Chemically-induced diabetes in this rat model was monitored by a glycemic check in both groups, taking daily blood samples from the caudal vein. Consumption of water and variation in bodyweight were also recorded. Rats treated with streptozotocin demonstrated a marked increase in daily water consumption up to 150–200 (mean 163.99 ± 1.9) mL/day 21 days after injection, while control values were 20–23 mL/day. Streptozotocin-treated diabetic rats reached glycemiclevels of 250–350 (mean 301.87 ± 4.1) mg/dL 21 days after injection whereas the nondiabetic rats had glycemic levels of 110–130 mg/dL. Treatment with streptozotocin also interrupted growth (see Figure 1). At 21 days after treatment with streptozotocin, all rats in Group A received an intravitreal transplant of human placental stem cells. Rats in Group B (sham-operated) were not treated, and were just monitored for water consumption, glycemia index, and bodyweight changes, in order to control the evolution of type 2 diabetes. The cell populations we transplanted were representative of human placental stem cells, ie, having elevated staminality, and were isolated from healthy placental tissue at term. We observed and studied the in vitro potential of these cell populations to differentiate into retinal phenotypes, with the aim of evaluating the differentiation potential of these cells in a diabetic animal model. In our study, we injected adult mesenchymal stem cells into the vitreous of the diabetic rats. Adult mesenchymal stem cells have a vast differentiation potential, with suppressive immunomodulatory activity via expression of immunomodulatory molecules, such as human leukocyte antigen G and interleukin-10. Moreover, adult mesenchymal stem cells were capable of inserting into rat tissue without inducing rejection phenomena. The cell populations were tagged with fluorescent markers in order to differentiate between them for in vivo transplantation. The markers we used were carbocyanine dyes with hydrophilic/hydrophobic characteristics. These dyes are usually used in living tissues and cells because they insert into the membrane and diffuse rapidly, staining the entire cell surface. We used 3,3′-dioctadecyloxacarbocyanine and 1-1′-dioctadecyl – 3,3,3′,3′ tetramethy 1 indodicarbocyanine. The advantages of these dyes are their manageability and thermal stability. Release of insulin in response to elevated glucose concentrations was tested in vitro using radioimmunoassay. One week before treating the animals, in vitro expansion of the human placental stem cells was carried out in culture. This procedure allowed us to maintain a stable phenotype with a high proliferative capacity. Characterization of the human placental stem cells and their differentiating potential was then performed by cytofluorimetric analysis. The profile of genetic expression was also studied in relation to nerve growth factor, brain-derived neurotrophic factor, ciliary-derived neurotrophic factor, glial-derived neurotrophic factor, and basic fibroblast growth factor.12,13 The day before intravitreal transplant, the human placental stem cells were trypsinized with 1 mL of trypsin, after aspiration of the culture medium and having washed the cells with about 3 mL of phosphate-buffered saline. After approximately 1 minute, the cells began to detach and were then stained with 3,3′-dioctadecyloxacarbocyanine fluorescent dye and resuspended in serum-free medium supplemented with epidermal growth factor and basic fibroblast growth factor at a concentration of 50,000 cells/μL. The cells were kept on ice for 4 hours before transplanting. Before receiving the transplant, Equithesin® was administered to all diabetic rats in Group A for general anesthesia.14 Equithesin was prepared according to the doses reported in Table 1, adhering to the order of components as listed. Before adding each successive component, a check was performed to ensure that each preceding component was well dissolved. The dose administered was 0.4 mL/100 g, adjusted for the weight of each individual rat. Before the surgical procedure, each rat was settled on a stereotactic table, and a single drop of iodopovidone 10% was instilled into the conjunctival sac to disinfect the periorbital and periocular skin, thereby preventing onset of endophthalmitis. After 5 minutes, another drop of iodopovidone 5% was instilled into the eye. When performing the surgical procedure, a microinjector pump connected to a flow rate system was used to control the flux of human placental stem cells, so that a maximum concentration of stem cells could be reached in the vitreous. The flow rate was maintained at 50,000 cells/μL. Intravitreal injection of the resuspended human placental stem cells was carried out via the pars planaat the level of the inferotemporal quadrant, 1 mm from the limbus. A drop of iodopovidone 5% was instilled in the operated eye after the intravitreal stem cell implant. The animals were monitored during postoperative reawakening and daily in the 7-day postsurgical period, to be able to identify any signs of suffering as early as possible. Postoperative discomfort was minimal. Evaluation of the diabetic rats in group B (sham operated) was also performed; the same surgical procedure was carried out in these rats using 0.2 μL physiological solution only. Immediately after the intravitreal transplant, all the rats were started ontopical antibiotic prophylaxis using tobramycin eye drops, instilling one drop in each operated eye three times daily for 10 days postoperatively. On postoperative day 21, all the diabetic rats in Group A underwent fluorangiography examination for clinical evaluation of diabetic retinopathy. Diabetic rats in Group B underwent the same examination. Fluorescein was injected at the caudal level. We evaluated the diabetic retinopathy from a clinical point of view in order to assess the eventual stabilization/regression of diabetic retinopathy, paying particular attention to retinal neovascularization and other signs of disease. This evaluation demonstrated the presence of neovascularization, ischemic areas, and microaneurysms present in the retina of the Group B diabetic rats. Moreover, vitreous levels of anti-apoptotic growth factors were quantified by real-time polymerase chain reaction using an enzyme-linked immunosorbent assay kit for vitreous growth factors. Multiplex bead assays were used for measurement of different growth factors and cytokines, in particular levels of basic fibroblastic growth factor, nerve growth factor, brain-derived neurotrophic factor, ciliary-derived neurotrophic factor,15 and glial-derived neurotrophic factor in the vitreal chamber. The increase in the levels of these factors in rats with transplanted human stem cells underscore the potential neuroprotective effects against oxidative damage of high growth factor levels released from stem cells.16–18 Vitreous samples (0.2 μL each sample) were used undiluted and immediately, in order to avoid vitreous condensation and solidification. Real-time polymerase chain reaction was performed according to the manufacturer’s instructions. Because of the limited volume of some of the samples, a minimum of duplicate tests were undertaken for each assay. Saline was substituted for the vitreous implant in the negative controls.


Potential role of intravitreal human placental stem cell implants in inhibiting progression of diabetic retinopathy in type 2 diabetes: neuroprotective growth factors in the vitreous.

Scalinci SZ, Scorolli L, Corradetti G, Domanico D, Vingolo EM, Meduri A, Bifani M, Siravo D - Clin Ophthalmol (2011)

Variation in water consumption and glycemia after treatment with streptozotocin. Rats treated with streptozotocin demonstrated a notable increase in daily water consumption, up to values of 150–200 mL/day at 21 days after treatment (mean 163.99 ± 1.9 mL/day), while reference values were 20–23 mL/day. Rats treated with streptozotocin reached mean glycemic levels of 250–350 mg/dL at 21 days after treatment (301.87 ± 4.1 mg/dL) unlike the nondiabetic rats (standard values 110–130 mg/dL).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3104799&req=5

f1-opth-5-691: Variation in water consumption and glycemia after treatment with streptozotocin. Rats treated with streptozotocin demonstrated a notable increase in daily water consumption, up to values of 150–200 mL/day at 21 days after treatment (mean 163.99 ± 1.9 mL/day), while reference values were 20–23 mL/day. Rats treated with streptozotocin reached mean glycemic levels of 250–350 mg/dL at 21 days after treatment (301.87 ± 4.1 mg/dL) unlike the nondiabetic rats (standard values 110–130 mg/dL).
Mentions: This preliminary interdisciplinary multicenter study was supported by the University of Bologna and carried out by the Department of General Surgery and Organ Transplants at the University of Bologna in collaboration with the Department of Ophthalmology at the University of Naples, and with the support of the Department of Ophthalmology at the US Army Health Clinic Camp Darby of Livorno. This study was approved by the ethical committee at the Sant’Orsola-Malpighi Hospital, University of Bologna, and all procedures were carried out under an institutionally approved protocol in accordance with the ethical principles and standards of the Federation of European Animal Science Association, following the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the current legislation on preclinical research in Italy. All the regulations of the Centralized Veterinary Service of Bologna were respected. Fifteen young adult male syngenetic Lewis rats, weighing 180–200 g (mean 188.56 ± standard deviation [SD] 7.14 g) were included in this randomized preclinical study and were rendered diabetic using streptozotocin. Glycemia10 was set at 90–120 (mean 98.3 ± 3.4) mg/dLat baseline (time 0, beginning of the study). Animals which did not meet these requirements were excluded. All rats selected for study were further subdivided into two groups, ie, Group A and Group B. Group A (n = 12) underwent an intravitreal human placental stem cell implant, and Group B (n = 3) served as the control. The rats in both groups were rendered diabetic by a single intraperitoneal injection of streptozotocin11 65 mg/kg. Starting from the day after streptozotocin injection, evening glycemia was evaluated daily and rats with a glycemia index ≥250 mg/dL for 3 consecutive days were considered to have developed type 2 diabetes mellitus. Chemically-induced diabetes in this rat model was monitored by a glycemic check in both groups, taking daily blood samples from the caudal vein. Consumption of water and variation in bodyweight were also recorded. Rats treated with streptozotocin demonstrated a marked increase in daily water consumption up to 150–200 (mean 163.99 ± 1.9) mL/day 21 days after injection, while control values were 20–23 mL/day. Streptozotocin-treated diabetic rats reached glycemiclevels of 250–350 (mean 301.87 ± 4.1) mg/dL 21 days after injection whereas the nondiabetic rats had glycemic levels of 110–130 mg/dL. Treatment with streptozotocin also interrupted growth (see Figure 1). At 21 days after treatment with streptozotocin, all rats in Group A received an intravitreal transplant of human placental stem cells. Rats in Group B (sham-operated) were not treated, and were just monitored for water consumption, glycemia index, and bodyweight changes, in order to control the evolution of type 2 diabetes. The cell populations we transplanted were representative of human placental stem cells, ie, having elevated staminality, and were isolated from healthy placental tissue at term. We observed and studied the in vitro potential of these cell populations to differentiate into retinal phenotypes, with the aim of evaluating the differentiation potential of these cells in a diabetic animal model. In our study, we injected adult mesenchymal stem cells into the vitreous of the diabetic rats. Adult mesenchymal stem cells have a vast differentiation potential, with suppressive immunomodulatory activity via expression of immunomodulatory molecules, such as human leukocyte antigen G and interleukin-10. Moreover, adult mesenchymal stem cells were capable of inserting into rat tissue without inducing rejection phenomena. The cell populations were tagged with fluorescent markers in order to differentiate between them for in vivo transplantation. The markers we used were carbocyanine dyes with hydrophilic/hydrophobic characteristics. These dyes are usually used in living tissues and cells because they insert into the membrane and diffuse rapidly, staining the entire cell surface. We used 3,3′-dioctadecyloxacarbocyanine and 1-1′-dioctadecyl – 3,3,3′,3′ tetramethy 1 indodicarbocyanine. The advantages of these dyes are their manageability and thermal stability. Release of insulin in response to elevated glucose concentrations was tested in vitro using radioimmunoassay. One week before treating the animals, in vitro expansion of the human placental stem cells was carried out in culture. This procedure allowed us to maintain a stable phenotype with a high proliferative capacity. Characterization of the human placental stem cells and their differentiating potential was then performed by cytofluorimetric analysis. The profile of genetic expression was also studied in relation to nerve growth factor, brain-derived neurotrophic factor, ciliary-derived neurotrophic factor, glial-derived neurotrophic factor, and basic fibroblast growth factor.12,13 The day before intravitreal transplant, the human placental stem cells were trypsinized with 1 mL of trypsin, after aspiration of the culture medium and having washed the cells with about 3 mL of phosphate-buffered saline. After approximately 1 minute, the cells began to detach and were then stained with 3,3′-dioctadecyloxacarbocyanine fluorescent dye and resuspended in serum-free medium supplemented with epidermal growth factor and basic fibroblast growth factor at a concentration of 50,000 cells/μL. The cells were kept on ice for 4 hours before transplanting. Before receiving the transplant, Equithesin® was administered to all diabetic rats in Group A for general anesthesia.14 Equithesin was prepared according to the doses reported in Table 1, adhering to the order of components as listed. Before adding each successive component, a check was performed to ensure that each preceding component was well dissolved. The dose administered was 0.4 mL/100 g, adjusted for the weight of each individual rat. Before the surgical procedure, each rat was settled on a stereotactic table, and a single drop of iodopovidone 10% was instilled into the conjunctival sac to disinfect the periorbital and periocular skin, thereby preventing onset of endophthalmitis. After 5 minutes, another drop of iodopovidone 5% was instilled into the eye. When performing the surgical procedure, a microinjector pump connected to a flow rate system was used to control the flux of human placental stem cells, so that a maximum concentration of stem cells could be reached in the vitreous. The flow rate was maintained at 50,000 cells/μL. Intravitreal injection of the resuspended human placental stem cells was carried out via the pars planaat the level of the inferotemporal quadrant, 1 mm from the limbus. A drop of iodopovidone 5% was instilled in the operated eye after the intravitreal stem cell implant. The animals were monitored during postoperative reawakening and daily in the 7-day postsurgical period, to be able to identify any signs of suffering as early as possible. Postoperative discomfort was minimal. Evaluation of the diabetic rats in group B (sham operated) was also performed; the same surgical procedure was carried out in these rats using 0.2 μL physiological solution only. Immediately after the intravitreal transplant, all the rats were started ontopical antibiotic prophylaxis using tobramycin eye drops, instilling one drop in each operated eye three times daily for 10 days postoperatively. On postoperative day 21, all the diabetic rats in Group A underwent fluorangiography examination for clinical evaluation of diabetic retinopathy. Diabetic rats in Group B underwent the same examination. Fluorescein was injected at the caudal level. We evaluated the diabetic retinopathy from a clinical point of view in order to assess the eventual stabilization/regression of diabetic retinopathy, paying particular attention to retinal neovascularization and other signs of disease. This evaluation demonstrated the presence of neovascularization, ischemic areas, and microaneurysms present in the retina of the Group B diabetic rats. Moreover, vitreous levels of anti-apoptotic growth factors were quantified by real-time polymerase chain reaction using an enzyme-linked immunosorbent assay kit for vitreous growth factors. Multiplex bead assays were used for measurement of different growth factors and cytokines, in particular levels of basic fibroblastic growth factor, nerve growth factor, brain-derived neurotrophic factor, ciliary-derived neurotrophic factor,15 and glial-derived neurotrophic factor in the vitreal chamber. The increase in the levels of these factors in rats with transplanted human stem cells underscore the potential neuroprotective effects against oxidative damage of high growth factor levels released from stem cells.16–18 Vitreous samples (0.2 μL each sample) were used undiluted and immediately, in order to avoid vitreous condensation and solidification. Real-time polymerase chain reaction was performed according to the manufacturer’s instructions. Because of the limited volume of some of the samples, a minimum of duplicate tests were undertaken for each assay. Saline was substituted for the vitreous implant in the negative controls.

Bottom Line: Intravitreal injection of human mesenchymal stem cells has been shown to be effective in slowing the progression of diabetic retinopathy in an animal model of chemically induced diabetes mellitus.Twenty-two Lewis rats were treated with an intravitreal human mesenchymal stem cell microinjection.Increased intravitreal and retinal concentrations of neuroprotective growth factors in rats confirm the neuroprotective activity of human mesenchymal stem cells in diabetic retinopathy.

View Article: PubMed Central - PubMed

Affiliation: Glaucoma and Low Vision Study Center, Department of General Surgery and Organ Transplants, University of Bologna, Bologna. sergio.unibo@unibo.it

ABSTRACT

Background: Intravitreal injection of human mesenchymal stem cells has been shown to be effective in slowing the progression of diabetic retinopathy in an animal model of chemically induced diabetes mellitus. We studied changes in growth factor levels released from human mesenchymal stem cells in the vitreous cavity as well as changes in growth factor levels in host retinal neurons following intravitreal injection.

Methods: Twenty-two Lewis rats were treated with an intravitreal human mesenchymal stem cell microinjection. Determination of neurotrophic factors released by human mesenchymal stem cells in the vitreous was carried out using real-time polymerase chain reaction.

Results: Detectable levels of neurotrophic factors were identified postoperatively in the vitreous of all rats.

Conclusion: Increased intravitreal and retinal concentrations of neuroprotective growth factors in rats confirm the neuroprotective activity of human mesenchymal stem cells in diabetic retinopathy.

No MeSH data available.


Related in: MedlinePlus