In diabetes, retinal dysfunctions exist prior to clinically detectable vasculopathy, however the pathology behind these functional deficits is still not fully established. Previously, our group published a detailed study on the retinal histopathology of type 1 diabetic (T1D) rat model, where specific alterations were detected. Although the majority of human diabetic patients have type 2 diabetes (T2D), similar studies on T2D models are practically absent. To fill this gap, we examined Zucker Diabetic Fatty (ZDF) rats - a model for T2D - by immunohistochemistry at the age of 32 weeks. Glial reactivity was observed in all diabetic specimens, accompanied by an increase in the number of microglia cells. Prominent outer segment degeneration was detectable with changes in cone opsin expression pattern, without a decrease in the number of labelled elements. The immunoreactivity of AII amacrine cells was markedly decreased and changes were detectable in the number and staining of some other amacrine cell subtypes, while most other cells examined did not show any major alterations. Overall, the retinal histology of ZDF rats shows a surprising similarity to T1D rats indicating that despite the different evolution of the disease, the neuroretinal cells affected are the same in both subtypes of diabetes.

Diabetic retinopathy is a major cause of reduced visual acuity and acquired blindness. The aim of this work was to analyze functional and vascular changes in diabetic Meriones shawi (M.sh) an animal model of metabolic syndrome and type 2 diabetes. The animals were divided into four groups. Two groups were fed a high fat diet (HFD) for 3 and 7 months, two other groups served as age-matched controls. Retinal function was assessed using full field electroretinogram (Ff-ERG). Retinal thickness and vasculature were examined by optical coherence tomography, eye fundus and fluorescein angiography. Immunohistochemistry was used to examine key proteins of glutamate metabolism and synaptic transmission. Diabetic animals exhibited significantly delayed scotopic and photopic ERG responses and decreases in scotopic and photopic a- and b-wave amplitudes at both time points. Furthermore, a decrease of the amplitude of the flicker response and variable changes in the scotopic and photopic oscillatory potentials was reported. A significant decrease in retinal thickness was observed. No evident change in the visual streak area and no sign of vascular abnormality was present; however, some exudates in the periphery were visible in 7 months diabetic animals. Imunohistochemistry detected a decrease in the expression of glutamate synthetase, vesicular glutamate transporter 1 and synaptophysin proteins. Results indicate that a significant retinal dysfunction was present in the HFD induced diabetes involving both rod and cone pathways and this dysfunction correlate well with the morphological abnormalities reported previously. Furthermore, neurodegeneration and abnormalities in retinal function occur before vascular alterations would be detectable in diabetic M.sh.

In diabetic subjects, early visual functional alterations such as color vision deficiencies (CVDs) are known to precede clinically apparent diabetic retinopathy. Prominent photoreceptor outer segment degeneration and an increase in the number of retinal dual cones (co-expressing S- and M-opsins simultaneously) have been described in diabetic rat models, suggesting a connection with the development of CVDs. As cone opsin expression is controlled by thyroid hormones, we investigated the diabetic retina in association with thyroid hormone alterations.

Identified as a member of the secretin/glucagon/VIP superfamily, pituitary adenylate cyclase-activating polypeptide (PACAP1-38) has been recognized as a hormone, neurohormone, transmitter, trophic factor, and known to be involved in diverse and multiple developmental processes. PACAP1-38 was reported to regulate the production of important morphogens (Fgf1, Bmp4, Gdf3) through PAC1-receptor in the newborn rat retina. To follow up, we aimed to reveal the identity of retinal cells responsible for the production and secretion of Fgf1, Bmp4, and Gdf3 in response to PACAP1-38 treatment. Newborn (P1) rats were treated with 100 pmol PACAP1-38 intravitreally. After 24 h, retinas were dissected and processed for immunohistochemistry performed either on flat-mounted retinas or cryosections. Brn3a and PAC1-R double labeling revealed that 90% of retinal ganglion cells (RGCs) expressed PAC1-receptor. We showed that RGCs were Fgf1, Bmp4, and Gdf3-immunopositive and PAC1-R was co-expressed with each protein. To elucidate if RGCs release these secreted regulators, the key components for vesicle release were examined. No labeling was detected for synaptophysin, Exo70, or NESP55 in RGCs but an intense Rab3a-immunoreactivity was detected in their cell bodies. We found that the vast majority of RGCs are responsive to PACAP, which in turn could have a significant impact on their development or/and physiology. Although Fgf1, Bmp4, and Gdf3 were abundantly expressed in PAC1-positive RGCs, the cells lack synaptophysin and Exo70 in the newborn retina, thus unable to release these proteins. These proteins could regulate postnatal RGC development acting through intracrine pathways.

Acute lower extremity ischemia is a limb- and life-threatening clinical problem. Rapid detection of the degree of injury is crucial, however at present there are no exact diagnostic tests available to achieve this purpose. Our goal was to examine a novel technique - which has the potential to accurately assess the degree of ischemic muscle injury within a short period of time - in a clinically relevant rodent model. Male Wistar rats were exposed to 4, 6, 8 and 9 hours of bilateral lower limb ischemia induced by the occlusion of the infrarenal aorta. Additional animals underwent 8 and 9 hours of ischemia followed by 2 hours of reperfusion to examine the effects of revascularization. Muscle samples were collected from the left anterior tibial muscle for viability assessment. The degree of muscle damage (muscle fiber viability) was assessed by morphometric evaluation of NADH-tetrazolium reductase reaction on frozen sections. Right hind limbs were perfusion-fixed with paraformaldehyde and glutaraldehyde for light and electron microscopic examinations. Muscle fiber viability decreased progressively over the time of ischemia, with significant differences found between the consecutive times. High correlation was detected between the length of ischemia and the values of muscle fiber viability. After reperfusion, viability showed significant reduction in the 8-hour-ischemia and 2-hour-reperfusion group compared to the 8-hour-ischemia-only group, and decreased further after 9 hours of ischemia and 2 hours of reperfusion. Light- and electron microscopic findings correlated strongly with the values of muscle fiber viability: lesser viability values represented higher degree of ultrastructural injury while similar viability results corresponded to similar morphological injury. Muscle fiber viability was capable of accurately determining the degree of muscle injury in our rat model. Our method might therefore be useful in clinical settings in the diagnostics of acute ischemic muscle injury.

The purpose of this work was to evaluate a potentially useful animal model, Meriones shawi (M.sh)-developing metabolic X syndrome, diabetes and possessing a visual streak similar to human macula-in the study of diabetic retinopathy and diabetic macular edema (DME). Type 2 diabetes (T2D) was induced by high fat diet administration in M.sh. Body weights, blood glucose levels were monitored throughout the study. Diabetic retinal histopathology was evaluated 3 and 7 months after diabetes induction. Retinal thickness was measured, retinal cell types were labeled by immunohistochemistry and the number of stained elements were quantified. Apoptosis was determined with TUNEL assay. T2D induced progressive changes in retinal histology. A significant decrease of retinal thickness and glial reactivity was observed without an increase in apoptosis rate. Photoreceptor outer segment degeneration was evident, with a significant decrease in the number of all cones and M-cone subtype, but-surprisingly-an increase in S-cones. Damage of the pigment epithelium was also confirmed. A decrease in the number and labeling intensity of parvalbumin- and calretinin-positive amacrine cells and a loss of ganglion cells was detected. Other cell types showed no evident alterations. No DME-like condition was noticed even after 7 months. M.sh could be a useful model to study the evolution of diabetic retinal pathology and to identify the role of hypertension and dyslipidemia in the development of the reported alterations. Longer follow up would be needed to evaluate the potential use of the visual streak in modeling human macular diseases.

We used immunocytochemistry to determine the presence and topographical density distributions of rods, cones, and intrinsically photosensitive retinal ganglion cells (ipRGCs) in the four-striped field mouse (Rhabdomys pumilio) and the Namaqua rock mouse (Micaelamys namaquensis). Both species possessed duplex retinas that were rod dominated. In R. pumilio, the density of both cones and rods were high (cone to rod ratio: 1:1.23) and reflected the species' fundamentally diurnal, but largely crepuscular lifestyle. Similarly, the ratio of cones to rods in M. namaquensis (1:12.4) reflected its nocturnal lifestyle. Similar rod density peaks were observed (R. pumilio: ~84467/mm2; M. namaquensis: ~81088/mm2), but a density gradient yielded higher values in the central (~56618/mm2) rather than in the peripheral retinal region (~32689/mm2) in R. pumilio. Two separate cone types (S-cones and M/L-cones) were identified implying dichromatic color vision in the study species. In M. namaquensis, both cone populations showed a centro-peripheral density gradient and a consistent S- to M/L-cone ratio (~1:7.8). In R. pumilio, S cones showed a centro-peripheral gradient (S- to M/L-cone ratio; central: 1:7.8; peripheral: 1:6.8) which appeared to form a visual streak, and a specialized area of M/L-cones (S- to M/L-cone ratio: 1:15) was observed inferior to the optic nerve. The number of photoreceptors per linear degree of visual angle, estimated from peak photoreceptor densities and eye size, were four cones and 15 rods per degree in M. namaquensis and 11 cones and 12 rods per degree in R. pumilio. Thus, in nocturnal M. namaquensis rods provide much finer image sampling than cones, whereas in diurnal/crepuscular R. pumilio both photoreceptor types provide fine image sampling. IpRGCs were comparably sparse in R. pumilio (total = 1012) and M. namaquensis (total = 862), but were homogeneously distributed in M. namaquensis and densest in the dorso-nasal quadrant in R. pumilio. The adaptive significance of the latter needs further investigation.

A thinning of the inner retina is one of the earliest potential markers of neuroretinal damage in diabetic subjects. The histological background is uncertain; retinal ganglion cell (RGC) loss and changes in the structure or thickness of the inner plexiform layer (IPL) have been suspected. Studies conducted on animal models on RGC pathology gave contradictory results. Hereby we present RGC numbers, distribution patterns and IPL thickness from Zucker Diabetic Fatty (ZDF) rats. After labelling RGCs on retinal whole mounts, isodensity maps were constructed, RGC numbers and distribution patterns analysed using a custom-built algorithm, enabling point-by-point comparison. There was no change in staining characteristics of the antibodies and no significant difference in average RGC densities was found compared to controls. The distribution patterns were also comparable and no significant difference was found in IPL thickness and stratification or in the number of apoptotic cells in the ganglion cell layer (GCL). Our results provide a detailed evaluation of the inner retina and exclude major RGC loss in ZDF rats and suggest that other factors could serve as a potential explanation for inner retinal thinning in clinical studies. Our custom-built method could be adopted for the assessment of other animal or human retinas.