Genetic analysis of cataract in Ihara epileptic rat.

We analyzed the mode of inheritance of cataract in the Ihara epileptic rat (IER) by crossing experiments, and mapped cataract-related genes by linkage analysis. Cataract did not develop in the F1 animals, but it developed in both male and female animals of backcross and F2. The occurrence rate of cataract was 48.5% in the backcross progeny and 19.4% in the F2 progeny. Thus, the character was considered to be inherited by the autosomal recessive mode. We found two groups that differed according to the time of onset among the backcross and F2 progeny: an early-onset group (EOG), in which cataracts developed by about 4 months after birth, and a late-onset group (LOG), in which cataracts developed 8 months or more after birth. Linkage analysis indicated the presence of one cataract gene each on Chromosome (Chr) 8 and Chr 15, and the cataract was demonstrated to be governed by more than one gene. The gene on Chr 8 was named Catil, and that on Chr 15. Cati2. Catil was involved in the occurrence of cataract, and the conditions required for cataract to develop were Cati1i/Cati1i or Cati1i/Cati1w. However, in the cataract rats with Cati1i/ Cati1w, the allele of Cati2 was always Cati2i/Cati2i. Cati2 was involved in the timing of onset of the cataract, and the precondition for early onset was Cati2i/Cati2i.

Immunohistochemical characterization of glomerular PDGF B-chain and PDGF beta-receptor expression in diabetic rats.

Platelet-derived growth factor (PDGF) was found to contribute to the pathophysiological process in the development and progression of glomerulosclerosis characterized by mesangial cell proliferation and accumulation of extracellular matrix. To examine the role of PDGF in the development of diabetic nephropathy, we conducted immunohistochemical analysis for PDGF B-chain (PDGF-B) and PDGF beta-receptor (PDGFR-beta) in the glomeruli of streptozotocin-induced diabetic rats. At 2, 4, and 12 weeks after the onset of diabetes, the expression of PDGF-B in glomeruli of diabetic rats was increased significantly as compared to control or diabetic rats treated with insulin. Similar changes were observed on PDGFR-beta immunostaining. The immunostaining of mirror sections revealed the existence of PDGF-B or PDGFR-beta not only in mesangial cells but also in visceral epithelial cells. Glomerular volume was significantly increased in diabetes. This early glomerular abnormality was prevented by an inhibition of PDGF system with trapidil as well as by the treatment of insulin. Our results suggest that the activation of the PDGF system in glomerular cells might play an important role in the development of early glomerular lesion in diabetes.

Role of PDGF B-chain and PDGF receptors in rat tubular regeneration after acute injury.

Various polypeptide growth factors are generally considered to be involved in the regulation of the nephrogenic process both after acute renal injury and during renal development. Because platelet-derived growth factor B-chain (PDGF-B) has been reported to be expressed in immature tubulus of the developing kidney, PDGF-B could play a role in the process of tubulogenesis. We examined the expression of PDGF-B and PDGF receptors alpha and beta and their localization in kidneys after ischemia/reperfusion injury. The mRNA expressions of PDGF-B, PDGFR-alpha, and PDGFR-beta were enhanced after injury. In the immunohistochemical analysis and/or in situ hybridization, PDGF-B and PDGFR-alpha, beta were expressed after reperfusion in the S3 segment of the proximal tubuli, where they were not expressed normally. The expressions of proliferating cell nuclear antigen and vimentin were concomitantly observed with PDGF-B and PDGFRs in the tubular cells of injured S3 segment at 48 hours after injury. Next, the inhibition of the PDGF-B/PDGFRs axis with either Trapidil or Ki6896, which was found to inhibit the phosphorylation of PDGFR-beta selectively, resulted in a rise of serum creatinine, higher mortality rate, abnormal regenerating process, and suppressed proliferation of tubular epithelial cells. These findings suggest that the PDGF-B/PDGFRs axis is involved in the proliferation of injured tubular cells and plays an important role in the regeneration of tubular cells from acute ischemic injury.

A mutation in the microtubule-associated protein tau in pallido-nigro-luysian degeneration.

We detected a missense mutation in exon 10 of tau that causes a substitution at codon 279 (N279K) in a Japanese patient with a familial background of parkinsonism and dementia originally described as pallido-nigro-luysian degeneration. This mutation is the same as one seen in a Caucasian family with pallido-ponto-nigral degeneration. The similarities between these two families suggest a common genetic mechanism that may account for the peculiar distribution of neuroglial degeneration with tauopathy.

Mossy fiber sprouting in the dentate gyrus in a newly developed epileptic mutant, Ihara epileptic rat.

We examined the correlation between seizure activity and development of mossy fiber sprouting in the hippocampal formation using Timm staining in a newly developed Ihara epileptic rat (IER). The sprouting of mossy fibers were clearly shown in the inner molecular portion of the dentate gyrus and in the stratum oriens of CA3 pyramidal cell layer with repeated seizures. A positive correlation between the frequency of generalized tonic and clonic convulsions and the Timm staining score in molecular layer of dentate gyrus was revealed. Sprouting of mossy fiber in IER seems to be linked with seizure activities resulting from epileptic bursts, not to the genetic mutation.

Expression of platelet-derived growth factor B-chain and beta-receptor in hypoxic/ischemic encephalopathy of neonatal rats.

Expression of platelet-derived growth factor B-chain and of its specific receptor (beta-receptor) was investigated in immature brains with hypoxic/ischemic injury. After the left common carotid arteries of seven-day-old rats were ligated and pups were placed in a hypoxic chamber, the protein and messenger RNA of both B-chain and beta-receptor were assessed using immunocytochemistry and northern analysis, respectively. Transcripts for B-chain were localized by in situ hybridization. Faint but definite expression of B-chain and beta-receptor was seen in the brains of untreated neonatal controls. Three to 48 h after hypoxia B-chain protein was generally increased above control levels, but focally decreased expression was seen in infarcted areas. Enhanced induction of messenger RNA of B-chain was seen in the both sides of cerebral cortices and hippocampi at 3 h. Strongly increased positivity for B-chain protein and mRNA occurred in the neurons surrounding the infarct. In situ hybridization still showed this up-regulation seven days after hypoxia. Beta-receptor protein expression was enhanced in some neurons immediately surrounding the infarct at 3 h of hypoxia, and marked up-regulation was seen at 16 h. Beta-receptor messenger RNA remained at control levels. Immunocytochemistry showed strong immunoreactivity for the beta-receptor on the neurons surrounding the infarct at 72 h. These results indicate that a neonatal hypoxic/ischemic insult induces neuronal up-regulation of the platelet-derived growth factor B-chain as well as beta-receptor immediately after hypoxia. While this up-regulation is relatively transient in most neurons, sublethal damage to neurons immediately surrounding an infarct induces sustained up-regulation. Through autocrine and paracrine mechanisms, platelet-derived growth factor B-chain molecules may act as a neuroprotective factor in immature brain experiencing with hypoxic/ischemic injury.

Nitric oxide synthase immunoreactivity related to cold-induced brain edema.

To determine the relationship between brain edema and the expression of nitric oxide synthase (NOS), we immunohistochemically studied the distribution and level of NOS in rat brain cold injury model. Vasogenic brain edema was produced by cortical freezing lesion. NOS immunohistochemical studies were performed 4 and 8 h, 1, 3, 5, 7, 14 and 21 days after injury. In control normotensive rats, immunoreactivity for NOS was observed in scattered neuronal cells as reported previously, but there was no reactivity in glial cells. In the present study in the cold injury model, however, fibrinogen staining showed extravasated plasma fluid extending to the white matter contralateral to the site of cold injury. NOS immunoreactivity was observed in most reactive astrocytes and a proportion of the microglial cells and macrophages in the white matter not only just beneath the area of cold injury but also in the contralateral side. The nerve cells in the edematous region scarcely showed additional immunoreactivity for NOS. The distribution of increased NOS relatively corresponded with the sites of extravasated plasma fluid demonstrated by fibrinogen staining. Electron microscopically, NOS was observed in astrocytes along the rough endoplasmic reticulum suggesting that NOS was produced in the cells and not taken up from the surroundings. Based on these findings, we postulate that brain edema and the simultaneously generated free radicals or some extravasated plasma components may induce expression of NOS in the reactive cells, and that the NO thus generated may be involved in the development of diffuse degeneration of the white matter which accompanies brain edema.

Neuronal and glial expression of heparin-binding EGF-like growth factor in central nervous system of prenatal and early-postnatal rat.

We investigated heparin-binding epidermal growth factor-like growth factor (HB-EGF) gene and protein expression in the central nervous system of prenatal and early postnatal rats. Assay by northern blot analysis showed that the HB-EGF mRNA was markedly expressed in the brain. In situ hybridization and immunohistochemical techniques showed that concordant expression of HB-EGF mRNA and protein was widely observed in the neurons and interfascicular oligodendrocytes, especially in the cerebellum, the hippocampus, the cerebral cortex, the subventricular area, and the brain stem nuclei. The intense expression of the HB-EGF mRNA was related anatomically and temporally to the proliferating neuroblasts in the external granular layer of the cerebellum and the subventricular layer of the cerebrum. These findings suggest that HB-EGF acts as a mitogen for the neuroblasts. Moreover, HB-EGF expression was observed in the post-mitogenic cells, such as in the cells of the molecular layer, the white matter, the IGL, or the Purkinje cells of the cerebellum. Since EGF receptors are abundantly expressed in the post-mitogenic period, the HB-EGF mRNA expression observed in the post-mitogenic period in our study suggests that HB-EGF also has a non-mitogenic function. These results suggest that HB-EGF significantly contributes to the development of the brain.

A rapid, easy technique for defining detailed anatomical orientation in pre-deparaffinized tissue sections.

During our previous histochemical studies on changes in anterograde and retrograde degeneration following optic nerve transection in rats, it was necessary to make brain sections in which various bilateral components of the visual system were symmetrically included. We discovered a rapid, easy technique that defines a detailed anatomical orientation in pre-deparaffinized tissue sections. The procedure is as follows: several drops of 1% toluidine blue solution are injected with a syringe under the paraffin section on the glass slide after the sections are cut and floated on warm water. After several seconds, the staining solution is removed, and the sections are then ready to be observed under a microscope. The anatomical orientation of the section can easily be defined without deparaffinization. By controlling the condition of sectioning procedures, we could obtain bilateral symmetrical paraffin sections containing all intended components of the visual system. This method is a rapid, simple and reliable way to get tissue sections which contain all of the multiple portions on the same plane.

Expression of platelet-derived growth factor after transient forebrain ischemia in the gerbil hippocampus.

The present study was conducted to clarify the role of platelet-derived growth factor-B chain (PDGF-B) in neuronal death after ischemia. Transient forebrain ischemia was induced in Mongolian gerbils by occluding the bilateral carotid arteries for 5 min. We investigated PDGF-B expression in the hippocampus after ischemia by immunohistochemistry, Northern blotting and in situ hybridization histochemistry. The results showed that PDGF-B is expressed in control CAI and CA3 neurons. In CA1, the amount of the PDGF-B transcript immediately increased, then disappeared 2 days after ischemia. Delayed neuronal death followed 1 day later. However, PDGF-B immunoreactivity in CA1 rapidly decreased and disappeared 12 h after transient forebrain ischemia, proceeding to delayed neuronal death. In contrast, the expression of both PDGF-B protein and the transcript was well preserved throughout the study in CA3, which remained viable even after ischemia. Accordingly, the selective neuronal susceptibility in the CA1 to ischemia corresponded with rapid disappearance of PDGF-B. PDGF-B expression may contribute to neuroprotective effect after ischemia.

Normal developing rat brain expresses a platelet-derived growth factor B chain (c-sis) mRNA truncated at the 5' end.

The 5' untranslated sequence (5' UTS) of platelet-derived growth factor B (PDGF-B/c-sis) mRNA is highly preserved through evolution, and inhibits translation of downstream coding sequences. In this study, using Northern analysis we identified two PDGF-B/c-sis mRNAs (3.5 kb and 2.6 kb) expressed in normal developing rat brain. In contrast to the constitutive expression of 3.5 kb mRNA, the expression of 2.6 kb mRNA increased markedly in accordance with those stages of brain development at which we had previously demonstrated an increased immunoreactivity for PDGF-B/c-SIS in neurons (Sasahara et al., 1992). By PCR cloning and the RNase protection assay, we determined the complete sequence of rat PDGF-B/c-sis, and found that the 2.6 kb transcript was a form of the 3.5 kb message truncated at the 5' end, and that the predominant 2.6 kb mRNA commenced 15 nt upstream of the signal peptide. Accordingly, it is suggested that the truncation of 5' UTS contributes to the expression of PDGF-B/c-SIS protein in the CNS. Lack of translational inhibitory 5' UTS of PDGF-B/c-sis transcript and resultant efficient protein translation have been reported in only a few transformed cells and cultured umbilical vein endothelial cells. We have extended this knowledge to the developing rat brain, and suggest that a similar mechanism could operate widely in non-transformed tissue in vivo.

Expression of heparin-binding epidermal growth factor-like growth factor in rat brain.

According to a recent report, messenger RNA coding for a member of the epidermal growth factor (EGF) family, heparin-binding EGF-like growth factor (HB-EGF), is expressed in the central nervous system (CNS). To obtain information about the role of HB-EGF in the brain, we carried out Northern analysis, in situ hybridization, and immunohistochemical studies evaluating the distribution and amounts of the growth factor using cDNA HB-EGF probes and an antibody raised against synthetic HB-EGF propeptide. Northern analysis revealed transcripts for HB-EGF in all regions of normal rat brain. Immunohistochemically, HB-EGF was demonstrated extensively in neurons at levels varying according to location. HB-EGF mRNA also was detected in neurons, suggesting that the growth factor is produced in these cells. HB-EGF mRNA and immunoreactivity were also demonstrated in interfascicular oligodendrocytes. These findings suggest that HB-EGF is a physiologic ligand for brain EGF receptors, and is likely to be important in neural function.

Apoptosis of medial smooth muscle cells in the development of saccular cerebral aneurysms in rats.

BACKGROUND AND PURPOSE: Using an animal model, we examined the role of apoptosis in the disappearance of medial smooth muscle cells (SMCs) during the development and growth of cerebral aneurysms. METHODS: Various degrees of cerebral aneurysms were induced in the right anterior cerebral artery-olfactory artery bifurcations in 65 Sprague-Dawley rats with ligation of the left common carotid artery and renal hypertension. We performed in situ end labeling of fragmented DNA with the lesions in 45 rats and electron microscopic study in the other 20 rats. RESULTS: With in situ end labeling of fragmented DNA, 4+/-3 apoptotic medial SMCs were detected in 35 of the 45 bifurcations. Apoptotic SMCs appeared in the medial layer in the "preaneurysm" group, the site speculated to show an aneurysmal change in the near future (6+/-3), and in the media in the "early aneurysm" group, which showed characteristics such as a small depression (5+/-3). In the "progressive aneurysm" group, they appeared more frequently at the aneurysmal neck (3+/-2) than the dome (1+/-1). By electron microscopic study, shrunken medial SMCs exhibiting morphological apoptotic changes such as chromatin condensation and fragmentation of the cytoplasm and nucleus were observed in the preaneurysm and early aneurysm groups and at the neck portion in the progressive aneurysm group. In the aneurysmal dome, SMCs showed late characteristics of apoptosis such as more advanced nuclear and cytoplasmic condensation and formation of apoptotic bodies. CONCLUSIONS: The present findings indicate that there is an association between apoptosis of medial SMCs and the formation of saccular cerebral aneurysms.

Platelet-derived growth factor B-chain expression in the rat retina and optic nerve: distribution and changes after transection of the optic nerve.

To test the possible involvement of platelet-derived growth factor B-chain (PDGF-B) in anterograde and retrograde degenerations of the CNS neurons, we studied the changes of PDGF-B localization and its mRNA expression in the rat retina and optic nerve (ON) after unilateral ON transection, using immunohistochemistry and in situ hybridization. In the control retinas immunoreactivity for PDGF-B and its mRNA expression were localized in the retinal ganglion cells (RGCs) and the nerve fiber layer. After ON transection PDGF-B immunoreactivity in the nerve fiber layer started to decrease on post-injury day 3 or 4. Atrophic changes in the RGCs started on day 5 just after the decrease of PDGF expression, and thereafter the RGC number decreased. In the longitudinal section of the ON rostral to the transected site, swollen axons showed intense PDGF-B immunoreactivity and macrophages, and some glial cells revealed a significant increase in both immunoreactivity and hybridization signals. Based on these findings, we hypothesized that the decrease in PDGF-B in RGCs after axotomy causes the loss of RGCs, and that increased PDGF-B expression in the ON plays a role in the cascade of tissue reactions following ON transection.

High performance seizure-monitoring system using a vibration sensor and videotape recording: behavioral analysis of genetically epileptic rats.

A new seizure-monitoring apparatus containing a piezoceramic vibration sensor combined with videotape recording was developed. Behavioral analysis of Ihara's genetically epileptic rat (IGER), which is a recently developed novel mutant with spontaneously limbic-like seizures, was performed using this new device. Twenty 8-month-old male IGERs were monitored continuously for 72 h. Abnormal behaviors were detected by use of a vibration recorder, and epileptic seizures were confirmed by videotape recordings taken synchronously with vibration recording. Representative forms of seizures were generalized convulsions and circling seizures. Generalized convulsions were found in 13 rats, and circling seizures in 7 of 20 animals. Two rats had generalized and circling seizures, and two rats did not have seizures. Although there was no apparent circadian rhythm to the generalized seizures, circling seizures occurred mostly between 1800 and 0800 h. A correlation between the sleep-wake cycle and the occurrence of circling seizures seems likely. Without exception, all the seizure actions were recorded by the vibration recorder and the videotape recorder. To eliminate the risk of a false-negative result, investigators scrutinized the information obtained from the vibration sensor and the videotape recorder. The newly developed seizure-monitoring system was found to facilitate detailed analysis of epileptic seizures in rats.

Distribution of heparin-binding EGF-like growth factor protein and mRNA in the normal rat kidneys.

Heparin-binding EGF-like growth factor (HB-EGF), a newly discovered potent mitogen and chemoattractant for smooth muscle cells, is a member of the EGF superfamily and binds to EGF receptors. To investigate the role of HB-EGF in the kidney, we determined the distribution of HB-EGF immunohistochemically in normal rat kidneys. The localization of mRNA expression was also studied by in situ hybridization, using a synthesized digoxigenin-labeled anti-sense riboprobe of HB-EGF. Immunohistochemical and in situ hybridization studies revealed that the tubular epithelial cells of the S3 segment of the outer stripe in the outer medulla were the predominant renal source of HB-EGF. In addition, in the immunohistochemical analysis, HB-EGF was ubiquitously present in the epithelial cells of the proximal tubules and the arterial smooth muscle cells, while HB-EGF expression was not detected in other parts of the kidney, including the glomeruli. Although EGF receptors were found to be present in the proximal tubules as well as in the distal tubules and collecting ducts, EGF has not been found to be expressed in the proximal tubules. Therefore, the present results indicate that HB-EGF might be a ligand for EGF receptors in the proximal tubules and might play a role in the functions of proximal tubules.

Altered expression of src tyrosine kinase after axotomy.

Although most peripheral neurons show regenerative changes after axotomy, neurons of some nuclei show degenerative changes. To study the mechanisms responsible for such differences in neuronal reactions after axotomy, we investigated the changes of pp60c-src and phosphotyrosine (pTyr) in the dorsal motor nucleus of the vagus nerve and the hypoglossal nucleus after transection of each nerve in adult rats using immunohistochemistry. Polyclonal antibody against pp60v-src stained the cytoplasm of neurons homogeneously in both the severed and non-severed sides of the vagal and hypoglossal nuclei from days 1 to 56 after axotomy. On the other hand, the monoclonal antibody against pp60v-src (mAb327, which recognizes src homology 3 region in pp60src as an epitope) showed different immunoreactivities. No pp60c-src immunoreactivity was observed in neurons in either the vagal and hypoglossal nuclei of the non-severed side between days 1 and 56, whereas in the severed side of the hypoglossal nucleus intense pp60c-src immunoreactivity was observed along the plasma membrane of neurons from days 5 to 28 after axotomy. In contrast, in the severed side of the vagal nucleus pp60c-src immunoreactivity appeared along the plasma membrane of neurons on day 5 and remained until day 7. Conformational changes accompanying the activation of pp60c-src are suggested to cause differences in immunoreactivities of these antibodies. The expression (or activation) of pp60c-src in the neurons was stronger and longer lasting in the hypoglossal nucleus, which regenerates successfully after axotomy, than in the dorsal motor nucleus of the vagus nerve, which undergoes necrobiotic reaction. These results indicate that pp60c-src plays an important role in regeneration after axotomy.

Cerebral aneurysms arising at nonbranching sites. An experimental Study.

BACKGROUND AND PURPOSE: The origin and pathogenesis of cerebral aneurysms arising at nonbranching sites are not clear. Using our animal model to induce cerebral aneurysms in rats, we examined induced aneurysms that developed at nonbranching sites. METHODS: In 35 Sprague-Dawley rats, the left common carotid artery was ligated and renal hypertension was produced to induce cerebral aneurysms. Twelve months later, the circle of Willis was carefully examined under a dissecting microscope. RESULTS: Other than cerebral aneurysms at branching sites of the circle of Willis, aneurysmal bulges developing at nonbranching sites were found in the proximal portion of the posteriorice rebral artery (P1) on the side of carotid ligation, which supposedly acted as a major collateral pathway after the ligation, in 19 of 35 treated rats. A total of 30 lesions were found in these 19 rats, and they were classified into fusiform aneurysms (22 lesions) involving the entire vessel wall for a short distance and saccular aneurysms (8 lesions) involving only a part of the wall and expanding laterally from the vessel wall. These P1s became larger in caliber and more tortuous after ligation. Aneurysms developed more frequently in proportion to these changes in these vessels. Moreover, most aneurysms in these vessels developed at or near the curvatures. All of the lateral aneurysms were found on the lateral wall of the curvatures of the vessels. CONCLUSIONS: The present findings indicate that cerebral aneurysms at nonbranching sites and saccular aneurysms at branching sites can occur under the same etiologic conditions. The site of origin is strongly related to hemodynamic stress.

Induction of platelet-derived growth factor beta-receptor in focal ischemia of rat brain.

Our previous study on the ischemia-induced expression of platelet-derived growth factor (PDGF)-B chain in the rat brain prompted us to examine expression of PDGF beta-receptor in the ischemic brain. Focal ischemia was induced by permanent tandem occlusion of middle cerebral and common carotid arteries in spontaneously hypertensive rats. Northern analysis revealed that ischemia significantly increased expression of the receptor in the ischemic neocortex at 4 and 7 days (328 +/- 109%; 323 +/- 119%, respectively, over control: n = 4, p < 0.05 versus sham). Neurons in infarct transiently showed increased immunostaining for the receptor at 1 day, whereas neurons in periinfarct area showed sustained and increased immunoreactivity from 1 to 14 days post-ischemia. Reactive glial cells in the external capsule and in molecular layer of the neocortex adjacent to infarct possessed enhanced immunoreactivity from 1 to 21 days. Furthermore, marked immunoreactivity was observed on brain macrophages in infarct and on the abluminal side of capillaries surrounding infarct from 4 to 7 days. These results demonstrated that ischemic insult increases expression of the PDGF beta-receptor at both the mRNA and protein level in the brain, suggesting its important role in cellular cascade of the ischemic brain.

Altered nitric oxide synthase immunoreactivity in the brain of stroke-prone spontaneously hypertensive rats.

To obtain information about the role of nitric oxide (NO) in the development of hypertensive cerebral lesions, we used immunohistochemical methods to study the distribution and level of nitric oxide synthase (NOS) in the brain of stroke-prone spontaneously hypertensive rats (SHRSPs). The early changes in the brain of SHRSPs were petechiae, edema and massive glial accumulation around fibrin deposits, which contained necrotized microvessels, whereas advanced cerebral lesions comprised massive bleeding, cavity formation and diffuse degeneration of the white matter. In the normotensive control rats, immunoreactivity for NOS was demonstrated in scattered neuronal cells, as has been reported previously, but there was no reactivity in glial cells. In the present study in SHRSPs, however, considerable NOS immunoreactivity was observed in most reactive astrocytes and in a proportion of the microglial cells and macrophages in the vicinity of the cortical lesions and in the subcortical white matter both ipsi- and contralateral to the cortical lesion. The nerve cells in the edematous region also showed weak immunoreactivity for NOS. The distribution of increased NOS in SHRSP brains corresponded well with the sites of extravasated plasma fluid as demonstrated by anti-fibrinogen antibody. Based on these findings, we postulate that edema and the simultaneously generated free radicals or some extravasated plasma components may induce expression of NOS in the reactive cells and nerve cells, and that the NO thus generated may be involved in the development of hypertensive cerebral lesions.