Study of postnatal development of the optic nerve and its vasculature in normal and streptozotocin-induced diabetic albion rat

Summary and ConclusionThe present study is mainly concerned with the study of the postnatal development of the optic nerve “representing the CNS” in diabetic and in control rats.To achieve this objectives diabetes mellitus was induced in neoborn Wistar rat pups by intraperitoneal injection of streptozotocin (STZ) at dose of “90 mg/Kg” for P2 rats. STZ injection produced tri-phasic change in the blood glucose level, first the blood glucose rose rapidly reaching a peak at P12 then rapidly fall down (initial phase of severe hyperglycemia). By the end of third week it returned to a level close to that of age matched controls and remained in normoglycemic state up to the seventh week, (phase of normoglycemia). After that it started to rise again gradually and steadily (phase of mild hyperglycemia).Complete computerized morphometric analysis was then applied to electron microscopic micrographs obtained from different age groups in both control and diabetic rats at different age groups during early and late phase of postnatal development and up to adulthood.It revealed the following results:• The optic nerve loses about 50 % of its total nerve fiber number during the first postnatal week. Loss of fibers then proceeds slowly over the next weeks. By P34 it reached less than 30 % of the total fiber number at P2. This process of elimination seems not affected by diabetic state except during the third week (severe hyperglycemic phase).• The myelinated axons were first detected at P8 in both control and diabetic rats. The number of myelinated axons gradually and significantly increased with age with a maximum rate between 2nd and 4th weeks. The number of myelinated axons was significantly decreased in diabetic animals compared to control in all examined ages.• A significant reduction in the thickness of myelin sheath in diabetic rats compared to age-matched controls was detected at all examined ages.• At P19 rats, the nodes of Ranvier were swollen and deformed in diabetic rats.• The rate of increase of whole cross-sectional area of optic nerve was invariably greater in the control than in the experimental groups at all examined ages.• The cross-sectional areas of both myelinted and non-myelinated axons were reduced significantly in the diabetic rats at all examined age compared to age matched controls.• Ultrastructural evidences of mitochondrial dysfunction, axonal degeneration, apoptosis of the glial and vascular cells and fissuring of myelin lamellae and complete lamellar disimpaction were common findings in the optic nerves obtained from diabetic rats.As inferred from these morphometric results, it is obvious that diabetes induced during the early postnatal period produced retardation in the myelination process of optic nerve axons.A question therefore arises. How could diabetes produce this retardation? To answer this question, another set of investigations was applied using immunohistochemical techniques to assess the process of “gliogenesis” which entail proliferation, migration and differentiation of oligodendroglial cells in the developing optic nerve.Three antibodies were applied to detect three cell stages in the course of oligodendrocyte differentiations; NG2 which detect oligodendrocyte progenitor cells (OPCs); O4 which detect premyelinating oligodendrocytes; and MBP which detect mature myelinating oligodendrocytes.At late stages of development it was rather difficult to count +cells in the field, so another quantitative assessment of the level of MBP was applied using “Western blotting technique” at P33 rats.The following results were obtained:• The number of “OPCs, (NG2+cells)”, premyelinating (O4+cells) and mature oligodendrocytes (MBP+cells) was reduced in the optic nerves in diabetic rats compared to age-matched controls.• Regarding the morphology, the oligodendroglial cells appeared less developed in diabetic rats compared to age-matched controls.• The concentration of MBP was reduced in diabetic rats compared to age-matched controls (as inferred from the density of the MBP bands in western blotting).A 2nd question arises from the previous results. Is the reduction in the number of oligodendroglial cells was a result of decreased cell proliferation or increased cell death or a combination of both? To answer this question, two experiments were done.The first was to assess the number of the proliferating cells using BrdU labeling and anti-BrdU antibodies to detect the cells incorporating that label. The second was to assess the number of dying cells by TUNEL technique which is designed to label the free 3’OH DNA termini in situ with chemically labeled and unlabeled nucleotides.The following results were obtained:• The number of BrdU+cells (proliferating cells) was much more decreased in diabetic rats compared to age-matched controls.• The number of TUNEL+cells (apoptotic cells) was much more increased in diabetic rats compared to age-matched controlsFrom these results, it could be concluded that:• Diabetes mellitus induced during the early postnatal period produced overall retardation in the growth and myelination of the optic nerve axons• The retardation in myelination of the optic nerve produced by neonatal-diabetes is attributed to the reduction in the number of myelinating cells (olilgodendrocytes)• Diabetes reduces the number of oligodendroglial cells both by a reduction in the rate of proliferation and by enhancement of the rate of cell death in the population of oligodendroglial cells.