Permeability changes in blood-retinal barrier of galactosemic rats are prevented by aldose reductase inhibitors.

Permeability-surface-area products (PA) for sucrose at the blood-retinal barrier (BRB) were determined with quantitative in vivo techniques and compared in control rats, rats fed a 50% galactosemic diet, and rats fed a diet containing both galactose and the aldose reductase inhibitor sorbinil. The mean PA +/- SE for controls was 0.656 x 10(-5) +/- 0.13 ml.g-1.s-1 and increased by approximately 500% in galactose-fed animals to 3.13 x 10(-5) +/- 0.32 ml.g-1.s-1. Animals fed both galactose and sorbinil showed no significant difference from control animals (P greater than .05), with a PA of 0.91 x 10(-5) +/- 0.22 ml.g-1.s-1. No breach in the BRB to horseradish peroxidase was detected in any of the groups. These results demonstrate an increased permeability at the BRB to small molecules in galactosemic rats that is prevented by an aldose reductase inhibitor. This suggests that the retinal capillary basement membrane thickening seen in galactosemic rats is associated with an increased permeability of the BRB and that aldose reductase is implicated in its pathogenesis.

The accessory optic system of rodents: a whole-mount HRP study.

The three-dimensional fiber pathways of the accessory optic system in three species of rodents (rat, golden hamster, guinea pig) were examined on whole-mounted preparations of the diencephalon and the midbrain, without sectioning, by anterograde labeling of retinal axons with horseradish peroxidase (HRP). HRP histochemical studies on the serial coronal sections were also done. In this study, only the accessory optic system on the side contralateral to the eye injection of HRP was clearly detected. The rat accessory optic system consisted of the inferior fasciculus, the superior fasciculus, the medial terminal nucleus, the lateral terminal nucleus, and the dorsal terminal nucleus. After the inferior fasciculus arrived at the ventromedial border of the cerebral peduncle, some fibers from the inferior fasciculus ran caudally to the medial terminal nucleus. The remaining fibers from the inferior fasciculus further proceeded dorsocaudally on the surface of the cerebral peduncle and left the inferior fasciculus at various levels of the cerebral peduncle to be mixed up with the fibers from the superior faciculus. The golden hamster accessory optic system also consisted of the inferior fasciculus, the superior fasciculus, the medial terminal nucleus, the lateral terminal nucleus, and the dorsal terminal nucleus. However, all fibers of the inferior fasciculus ran caudally on the lateral surface of the hypothalamus or along the ventromedial border of the cerebral peduncle to terminate at the medial terminal nucleus. The guinea pig accessory optic system and rat accessory optic system were similar, but the posterior fibers of the superior fasciculus decreased in number, and the dorsal terminal nucleus and the posterior portion of the lateral terminal nucleus were not observed in the guinea pig accessory optic system.

Prevention of pericyte ghost formation in retinal capillaries of galactose-fed dogs by aldose reductase inhibitors.

A distinguishing feature of early diabetic retinal vascular changes is the selective degeneration of pericytes (mural cells) from the retinal capillary vessels. Loss of these pericytes has been proposed to be associated with decreased capillary tonicity, the formation of microaneurysms, and vessel dilation. The role of aldose reductase in the progression of diabetic retinopathy has been investigated in age- and sex-matched beagles fed a 30% galactose diet with or without the aldose reductase inhibitors sorbinil or M79175. Eyes were periodically enucleated from dogs in each group and their retinal capillaries were examined as trypsin-digested flat preparations. Before the clinical appearance of retinal changes, pericyte ghost formation was observed in the eyes of three fourths of the dogs after 21 months and all of the dogs after 24 months of galactose feeding. Many of the capillaries containing pericyte ghosts demonstrated an apparent proliferation of endothelial cells and acellular vessels. No pericyte ghosts or abnormal findings were observed in retinas from either normal control (zero of nine) or galactose-fed dogs treated with aldose reductase inhibitors (zero of 16). These findings indicate that aldose reductase inhibitors can prevent the formation of pericyte ghosts and other subsequent capillary changes in experimental retinopathy.

Morphological survey of neurotensin-like immunoreactive neurons in the hypothalamus.

Neurotensin-like immunoreactive neuronal perikarya, fibers and terminals in in the rat hypothalamus were investigated by light and electron microscopic immunocytochemistry. Distributional density and pattern of these elements were clarified. Fine structure of immunoreactive neuronal perikarya with respect to development of cell organellae and immunoreactive dense granules was also elucidated. Features of immunoreactive processes, dendrites and preterminal axons were examined electron microscopically. In addition to the above findings by light and electron microscopic immunocytochemistry, we examined the coexistence of dopamine and neurotensin-like immunoreactive substances in these same neurons in the arcuate and periventricular nuclei. This was proved by the application of fluorescence histochemistry and immunocytochemistry on the same sections. Moreover, we speculated that the ascending noradrenergic neurons influence the neurotensin immunoreactive neurons in the paraventricular nucleus since a marked decrease in the number of neurotensin-like immunoreactive neuronal perikarya was observed after transection of ascending noradrenergic pathway.

Time of vasopressin neuron origin in the mouse hypothalamus: examination by combined technique of immunocytochemistry and [3H]thymidine autoradiography.

A time-course study on production of vasopressin neurons (VP-neurons) in the mouse hypothalamus was carried out by application of [3H]thymidine autoradiography and PAP-immunocytochemistry simultaneously on the same tissue sections. In the paraventricular nucleus (PVN) and the supraoptic nucleus (SON), heavily [3H]thymidine-labeled VP-neurons were detected only in the specimens of the animals exposed to the isotope on the gestational day 12. While in the suprachiasmatic nucleus (SCN), heavily isotope-labeled VP-neurons were observed in the specimens of the animals exposed to the isotope on the gestational day 12 or day 14. Therefore, the production of the VP-neurons in the SCN is prolonged and slightly more delayed than that in the SON or PVN.

Coexistence of dopamine and neurotensin in hypothalamic arcuate and periventricular neurons.

The coexistence of dopamine and neurotensin in the same neuronal perikarya in the arcuate nucleus of the rat hypothalamus was examined by combined fluorescence histochemistry and immunohistochemistry on the same tissue sections and we obtained the evidence of the coexistence of two substances. The functional significance of those two substances for the prolactin release from the anterior pituitary was also briefly discussed.

Hypothalamo-retinal centrifugal projection in the dog.

Horseradish peroxidase (HRP)-filled neurons and their processes were consistently detected in the ventral portion of the dog hypothalamus after intraocular injection of HRP. The number of HRP-filled neurons decreased in parallel with the extent of the resection of the optic nerve. HRP-filled neurons were never detected in specimens with a complete resection of the optic nerve. These findings strongly indicate that these HRP-filled neurons in the ventral hypothalamus are the source of centrifugal fibers to the retina.

Vasoactive intestinal peptide (VIP)-like immunoreactive neurons located in the rat suprachiasmatic nucleus receive a direct retinal projection.

The existence of a direct projection from retinal ganglion cells to vasoactive intestinal peptide (VIP)-like immunoreactive neuronal elements in the rat suprachiasmatic nucleus (SCN) was revealed by combining analysis of degenerating axons following enucleation and electron microscopic immunocytochemistry. Degenerating axons appeared to make synaptic contact with VIP-like immunoreactive dendrite and neuronal perikarya in the ventral part of the SCN. The possibility of neuronal input from retinal ganglion cells to axons of VIP-like immunoreactive neurons was also suspected since axo-axonic synapses were detected between degenerating axons and axons with VIP-like immunoreactivity. Thus, VIP-like immunoreactive neurons in the SCN receive several neuronal inputs, including those from the retina, and may play a significant role in circadian entrainment.

Postnatal development of vasoactive intestinal polypeptide immunoreactive amacrine cells in the rat retina.

The ontogenic development of vasoactive intestinal polypeptide (VIP)-immunoreactive amacrine cells in the rat retina was studied using peroxidase-antiperoxidase (PAP) immunohistochemistry. In the rat retina, VIP immunoreactivity appeared entirely in postnatal stage. On the 12th postnatal day, VIP-immunoreactive amacrine cells could be first detected. However, VIP immunoreactivity was very weak and VIP-immunoreactive amacrine cell processes could not be observed at this stage. On the 21st postnatal day, VIP-immunoreactive amacrine cell bodies became more mature and their processes were distinctly observed, resembling their appearance in adult rat retinas. VIP immunoreactivity could be detected in both stratified and diffuse amacrine cells at this stage. The relationship between the first appearance of VIP-immunoreactive amacrine cells and the synaptic formation in the inner plexiform layer is briefly discussed.

An immunohistochemical investigation of vasoactive intestinal polypeptide in the colon of patients with Hirschsprung's disease.

The distribution of vasoactive intestinal polypeptide (VIP) in the colon of patients with Hirschsprung's disease was investigated by the peroxidase-antiperoxidase (PAP) immunohistochemical method. Three colonic segments, ganglionic, oligoganglionic and aganglionic, were stained by the unlabeled antibody enzyme method. VIP immunoreactive nerve cell bodies, nerve fibers and nerve endings were distributed throughout the ganglionic and oligoganglionic segments. In contrast, the aganglionic segment contained no VIP nerve endings and the number of fibers was reduced. These differences are thought to be a cause of constriction of the colon in Hirschsprung's disease and VIP neurons are therefore believed to participate in the relaxation of smooth muscle.

Distribution of the VIP-like immunoreactive neurons in the cat central nervous system.

Immunohistochemical topographic localization of the vasoactive intestinal polypeptide (VIP)-like immunoreactive neurons in the cat brain was investigated using a peroxidase anti-peroxidase technique. VIP-like immunoreactive neurons were mainly localized in the cerebral cortex, limbic cortex, hypothalamic nuclei; suprachiasmatic nucleus, supraoptic nucleus, paraventricular nucleus, periventricular nucleus and arcuate nucleus, and in the midbrain; such as the central grey and the raphe nucleus. It was demonstrated that VIP-like immunoreactive neurons were widely distributed in the cat brain, particularly in the hypothalamus, compared with those of the rat and mouse; though whether these differences were species-related or due to differences in the physiological conditions remains to be determined. This is the first report of VIP neuronal perikarya in the arcuate nucleus of mammalian species, although these cells are present in the arcuate nucleus of birds.

The accessory optic system of the rabbit, cat, dog and monkey; a whole-mount HRP study.

The three-dimensional fiber pathways of the accessory optic system in the rabbit, cat, dog and monkey were studied in whole-mounted preparations of the diencephalon and the midbrain, without sectioning, by anterograde labeling of retinal axons with horseradish peroxidase (HRP). HRP histochemical studies on alternate serial coronal sections were also performed. The rabbit accessory optic system exhibited two fasciculi (the inferior fasciculus, and the superior fasciculus consisting of the anterior fibers and the posterior fibers) and three terminal nuclei (the medial terminal nucleus, and the anterior and posterior portions of the lateral terminal nucleus), but lacked the dorsal terminal nucleus. In the cat and dog, only the posterior fibers of the superior fasciculus were detected. The inferior fasciculus and the anterior fibers of the superior fasciculus were absent. The medial terminal nucleus and the posterior portion of the lateral terminal nucleus were commonly observed in the cat and dog. The cat accessory optic system possessed the dorsal terminal nucleus, and the dog accessory optic system possessed the anterior portion of the lateral terminal nucleus. The monkey (Macaca fuscata) accessory optic system consisted of the posterior fibers of the superior fasciculus, but blacked the inferior fasciculus and the anterior fibers of the superior fasciculus. Most of the posterior fibers terminated in the well-developed posterior portion of the lateral terminal nucleus located on the upper surface of the cerebral peduncle. A small number of posterior fibers projected to the poorly-developed medial terminal nucleus. Based on these findings, species differences in the mammalian accessory optic system were discussed.

A whole-mount horseradish peroxidase study of the retinal central projection in normal and monocular rats.

Anatomical mapping of the retinal central projection in albino rats was performed by whole-mount HRP histochemistry, after intraocular injection of HRP. The overall features of the retinal central pathways from the optic chiasma to the superior colliculus as well as the accessory optic systems in normal and monocular rats were clearly visualized, following application of a modified tetramethyl benzidine method on the whole-mounted brain stems including the diencephalon and the mid-brain. When combined with a partial retinal lesion, the whole-mount HRP method made feasible detection of the spatial retinotopy of the collicular projection.

Localization of vasoactive intestinal peptide (VIP) messenger RNA (mRNA) in amacrine cells of rat retina.

We detected vasoactive intestinal peptide (VIP) messenger RNA (mRNA) in the rat retina using an in situ hybridization technique and a 35S-labelled cDNA probe. VIP mRNA was present in the cells of the inner nuclear layer (INL). The VIP mRNA-positive cells showed a distribution similar to that of the VIP-like immunoreactive amacrine cells. This observation suggests that VIP mRNA undergoes transcription in the VIP-immunoreactive amacrine cells.

Aldose reductase localization in dog retinal mural cells.

The selective degeneration of retinal mural cells, a hallmark of early human diabetic retinopathy, has also been reported to occur in both diabetic and galactose-fed dogs. By employing antibodies raised against purified dog lens aldose reductase the presence of aldose reductase can be immunohistochemically demonstrated in the cytoplasm of mural cells but not endothelial cells of dog retinal vessels isolated by trypsin digestion. This immunohistochemical staining is similar to that observed with isolated human retinal vessels.

Immunohistochemical investigations of gut hormones in the colon of patients with Hirschsprung's disease.

The distributions of gut hormones in the colon of Hirschsprung's disease were investigated by the peroxidase-antiperoxidase (PAP) immunohistochemical method. Three colonic segments (ganglionic, oligoganglionic, and aganglionic) were stained by the unlabeled antibody enzyme method. The immunoreactivity of vasoactive intestinal polypeptide (VIP) was found to be reduced in the oligoganglionic and aganglionic segments. Antisera to substance P and met-enkephalin demonstrated immunoreactive cells and fibers in the ganglionic segment, whereas these cells and fibers were almost completely absent in the oligoganglionic and aganglionic segments. A similar distribution was seen for the mucosal endocrine cells with somatostatin immunoreactivity. Antisera to neurotensin, motilin, bombesin, and cholecystokinin revealed no immunoreactivity in the normal colon or the three segments. The differences in these peptides between normal and impaired colonal segments may be one of the causes of colon constriction in Hirschsprung's disease.

The effect of aldose reductase inhibitor, statil, on hyperpermeability of iridial vessels in experimental galactosemic rats.

Thirty young male Sprague-Dawley rats were divided into three groups: the control group, given a normal diet; the galactose group, given a 25% galactose diet; and the aldose reductase-galactose group, given a 25% galactose diet containing 0.046% Statil, an aldose reductase inhibitor. The results suggested that Statil could prevent hyperpermeability of iridial vessels in galactose-fed rats.

Proliferative changes of lens epithelial cells in rat and mouse galactose cataracts--examination using whole-mount preparations.

The proliferative activity of the lens epithelium in the early stages of cataract crisis was investigated in rats and mice using the 3H-thymidine autoradiographic method with the whole-mount preparations of total lens epithelial cells. Three-week-old Sprague-Dawley (SD) rats were given a diet which included galactose in three different concentrations (15%, 25%, 50%) to produce sugar cataracts of three different degrees. Seven-week-old ICR mice were given a 50% galactose-diet. In the lenses of control rats and mice, 3H-thymidine labeled cells were observed mainly in the germinative zone at the lens equator; a few labeled cells were detected in the anterior subcapsular central zone. In the lenses of the SD rats on the 4th day of the diet, labeled cells increased remarkably in the central zone. However, labeled cells decreased as the cataract progressed. The peak in the number of labeled cells was observed on the 4th day of the diet regardless of the galactose concentrations, and was not proportional to the degree of the cataracts. In the galactose-fed ICR mice, the blood galactose level was high, but there was no increase in the number of labeled cells or the development of galactose cataract. The marked increase of labeled cells in the central zone in the SD rat lenses had probably occurred because of the accumulation of galactitol.

[Differences in cataractogenesis according to neodymium-YAG laser injury site].

The author examined morphological changes of the lens induced by mechanical damage with a Q-switch Nd-YAG laser (anterior capsule, anterior subcapsular deep cortex, posterior capsule). In the ruptured anterior capsule group, epithelial cell proliferation covered the ruptured capsule, and separation of posterior subcapsular sutures and swelling of the posterior subcapsular end of lens fiber cells were observed. In the ruptured subcapsular deep cortex group, swollen lens fiber cells were observed in both the anterior subcapsular and the posterior subcapsular cortex. In the ruptured posterior capsule group, the ruptured cortex was not repaired. Swollen lens fiber cells were first recognized in areas surrounding the ruptured capsule, then at the posterior side of the equator, and finally at the anterior subcapsular cortex. The continuity of the lens capsule and lens fiber cells themselves is very important part to maintain lens clarity. The destruction of this feature induced swelling of fiber cells on the opposite side of the injury area.

[Movement of regenerated lens epithelial cells in 50% galactose cataract and an aldose reductase inhibitor].

In experimental galactose cataract of rats, lens fiber cells were gradually destroyed by swelling and liquefaction occurring due to the accumulation of galactitol. In addition to the destruction of lens fiber cells, marked regeneration of lens epithelial cells was universally observed. In this study, we used the technique of 3H-thymidine autoradiography and examined the movement of regenerated (DNA synthesis) lens epithelial cells. On the fourth day from the starting of 50% galactose chow, 3H-thymidine was injected into the anterior chamber. After one week, 3H-thymidine-labelled epithelial cells was observed at the bow region of the equatorial area. After 2 to 4 weeks, labelled cells were found in the regenerated lens fibers of the cortex. After 3 weeks, labelled cells about to be destroyed by swelling and liquefaction were recognized. However, a few labelled cells were still observed in the epithelial cell layer. The same experiments were performed in two groups of normal chow-fed one and 50% galactose + an aldose reductase inhibitor (Statil)-fed one. The movements of 3H-thymidine labelled epithelial cells in the above two groups were almost identical. In addition, the movement of labelled cells was normalized by an aldose reductase inhibitor.