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The purpose of this study is to evaluate the changes in neurofilament light (NF-L) protein in the optic nerve in rat kainate and monkey ocular hypertension models. In the rat model, optic nerve damage was induced by kainate injection into the vitreous body. In the monkey model, photocoagulation of the trabecular meshwork led to laser-induced ocular hypertension. NF-L in optic nerve extract was quantified by quantitative immunoblot using an imaging analyzer. The amount of NF-L in optic nerve was compared between normal and kainate-treated groups at 7 days after kainate injection. Specimens from rat optic nerves and retinas were evaluated histologically to examine the correlations between damage and amount of NF-L in the optic nerve. In monkeys, the amount of NF-L in the optic nerve was compared between control fellow and ocular hypertensive eyes. Injection of kainate induced morphological optic nerve and retinal damage in rats. The amount of NF-L in the optic nerve was significantly reduced in kainate-treated eyes (vs. normal eyes). The amount of NF-L correlated with the cell count in the ganglion cell layer and the axon number in the optic nerve at 7 days after kainate injection. Further, 6-cyano-7-nitro-quinoxaline-2,3-dione, a non N-methyl-D-aspartate receptor antagonist, suppressed the kainate-induced reduction in NF-L and retinal damage. In the monkey model, ocular hypertension and morphological optic nerve damage were shown by laser-treated eyes. The amount of NF-L in the optic nerve was reduced in laser-treated eyes. In conclusion, in rat kainate and monkey ocular hypertension models, immunoblot evaluation of NF-L shows the reduction of the amount of NF-L in the optic nerves of treated-eyes. The amount of NF-L correlated with the morphological retinal and optic nerve damage in rats. These findings indicate that immunoblot evaluation of NF-L in the optic nerve may provide a quantitative index of optic nerve damage.

We have analyzed the immunolabeling with the antibody RT97, a good marker for ganglion cell axons in several species, in the normal and regenerating visual pathways of teleosts. We have demonstrated that RT97 antibody recognizes several proteins in the tench visual system tissues (105, 115, 160, 200, 325 and 335 kDa approximately). By using immunoprecipitation and Western blot we have found that after crushing the optic nerve the immunoreactivity to anti RT97 increased markedly in the optic nerve. In immunohistochemical analysis we also found a different pattern of labeling in normal and regenerating visual pathways. In normal tench RT97 is a good marker for the horizontal cells in the retina, for growing ganglion cell axons which run along the optic nerve from the retina to the optic tectum and of the axon terminals in the stratum opticum and stratum fibrosum and griseum superficiale in the optic tectum. After optic nerve crush, no immunohistochemistry modifications were observed in the retina. However, in accordance with Western blot experiments, in the optic nerve intensely stained groups of regenerating axons appeared progressively throughout the optic nerve as far as the optic tectum. We conclude that the antibody RT97 is an excellent marker of growing and regenerating axons of the optic nerve of fish.

We have established a cell line (TB) from a cerebrospinal fluid (CSF) specimen of a patient with a primary leptomeningeal melanomatosis. TB cell line was immunoreactive with the antibodies for low molecular weight neurofilament protein, vimentin, neuron-specific enolase, chromogranin, synaptophysin and HMB-45 (an antibody sensitive and specific for melanoma). When TB cells were transplanted into nude mice, the same immunohistochemical pattern present in cultured cells was found but surprisingly, a positive staining for desmin was observed. Significant amounts of serotonin and its metabolite were detectable. Retinoic acid but not nerve growth factor was able to induce differentiation towards a neuronal phenotype. In summary, TB cells represent primitive neuroectodermal cells having the potential for neuronal, myoblastic and possibly melanoblastic differentiation., (Copyright 1999 Elsevier Science B.V.)

Serum amyloid P component (AP) and C-reactive protein (CRP) are normal serum components which belong to the pentraxin family of proteins. These proteins have been previously localized by immunohistochemical method to the brain lesions of Alzheimer's disease (AD). AP is a constant constituent of amyloid deposits including those found in AD. Both AP and CRP have been localized to AD neurofibrillary tangles. An indirect role for these proteins has been previously suggested in the etiology of AD. We studied the effects of serum AP and CRP on a human-derived neuronal cell line (hNT). In treated cell cultures, AP and CRP were detected immunohistochemically within hNT neurons, indicating cellular uptake of these proteins. Serum AP at the lowest serum physiological concentration (8 microgram/ml) showed a marked toxicity to hNT neurons. CRP also displayed toxicity to the hNT neurons but at a level compatible with inflammatory states (50 microgram/ml). These results suggest a more direct role for serum AP and CRP in the pathogenesis of AD., (Copyright 1998 Elsevier Science B.V.)

Immunohistochemical labelling with the monoclonal antibody SMI32 to non-phosphorylated epitopes on neurofilament proteins of high molecular weight class was low in rat central optic fibers of controls. After unilateral transection of optic nerve, a strong, transient increase of labelling with SMI32 occurred in degenerating fibers of optic tract at 2 and 4 days, which then declined at 8 and remained low at 21 days. Consequently, immunostaining with SMI32 may serve as a positive marker for degenerating fibers in rat optic system.

When grafting human mesencephalic tissue to patients suffering from Parkinson's disease, the number of surviving dopamine (DA) neurons in the graft is probably crucial. It may be possible to increase the number of DA neurons available for grafting to a patient by pooling tissue from many human embryos collected over several days or by obtaining more DA neurons from each embryo. We have addressed these issues by cryopreserving human mesencephalic DA neurons prior to transplantation and also by grafting human embryonic diencephalic DA neurons. The effects of cryopreservation were assessed 4-15 weeks after xenografting ventral mesencephalic tissue into the DA-depleted striatum of immunosuppressed rats with unilateral 6-hydroxydopamine lesions of the mesostriatal pathway. Control rats grafted with fresh mesencephalic tissue displayed robust reductions in amphetamine-induced turning following transplantation. Functional effects of the cryopreserved mesencephalic grafts were only observed in the one rat out of nine which contained the largest graft in this group. The number of tyrosine hydroxylase immunoreactive neurons in animals transplanted with cryopreserved tissue was significantly reduced to 9% of fresh tissue control grafts. Morphological analysis showed that cryopreserved DA neurons were approximately 22% and 28% smaller regarding the length of the long and short axis, respectively, when compared to the neurons found in fresh grafts. In the second part of the study, the survival and function of human embryonic diencephalic DA neurons were examined following xenografting into the DA-depleted rat striatum. A reduction of motor asymmetry was observed in two out of seven diencephalon-grafted rats. This finding was consistent with a good graft survival in these particular rats, which both contained large grafts rich in tyrosine hydroxylase immunoreactive neurons. Moreover, there was immunopositive staining for graft-derived fibers in the rat striatum containing tyrosine hydroxylase and human neurofilament, both in rats grafted with mesencephalic and diencephalic DA neurons. These findings suggest that cryopreservation, using the current technique, is not a suitable storage method for use in clinical trials of DA neuron grafting in Parkinson's disease. On the other hand, the application of alternative sources of DA neurons may in the future develop into a strategy which can increase the number of neurons obtainable from each human embryo.

Protein kinase C (PKC) and growth-associated protein-43 (GAP-43) were investigated immunohistochemically in the dorsal motor nucleus of the vagus nerve and the hypoglossal nucleus after axotomy using monoclonal antibodies against type I, II and III PKC and GAP-43. In the control side of both nuclei, anti-type I and II PKC weakly stained neuronal cell bodies, while anti-type III PKC did not show any reaction with neurons. In the axotomized side of both nuclei, anti-type II PKC antibody intensely stained affected nerve cell bodies as well as plasma membrane. Some of the severed neurons showed intensified reactions for both anti-type II PKC and anti-GAP-43 antibodies in the serial sections. These findings suggest that axotomy increases the type II PKC of the severed neurons, and type II PKC seems to phosphorylate some protein, such as GAP-43, and plays some role in the retrograde neuronal reaction.

Using two monoclonal antibodies, tau-1 and PHF-1, and a sequential staining method combining double-labeling immunofluorescence and Bielschowsky silver staining, we have demonstrated the presence of two populations of dystrophic neurites (DNs) with distinct immunocytochemical and argentophilic characteristics. Tau-1 and PHF-1 immunoreactivity were co-localized in many DNs. However, approximately 20% of the DNs were immunoreactive for PHF-1 only. PHF-1 single-labeled DNs were not visible or very weak with Bielschowsky silver stain. Of DNs continuous with neurofibrillary tangles (NFTs), tau-1/PHF-1 double-labeled DNs were continuous with intracellular NFTs only, while PHF-1 single-labeled DNs were continuous with extracellular NFTs only. Furthermore, the population of DNs that cluster around extracellular NFTs is different from those that cluster around or within senile plaques. The combined use of tau-1 and PHF-1 immunocytochemistry may provide a more accurate indication of the number of extracellular DNs and extracellular NFTs, which may aid in the diagnosis of severe and advanced AD cases.

Organotypic cerebellar cultures were exposed to the mitotic spindle inhibitor, vinblastine, and examined by light and electron microscopy, and electrophysiologically. Purkinje cells in exposed cultures had stunted, varicose dendrites when observed in preparations reacted with antibody to nonphosphorylated neurofilament protein. Ultrastructurally, microtubules were selectively reduced in Purkinje cell dendrites, which contained disarrayed excess smooth endoplasmic reticulum, but no large accumulations of densely packed neurofilaments. Spontaneous cortical discharges were initially increased in vinblastine treated cultures and then ceased, at which time the cultures became unresponsive to electrical stimulation. The vinblastine-induced changes were attributed to impaired intracellular transport secondary to disruption of Purkinje cell dendritic microtubules.

Using antibodies against parvalbumin and neurofilament triplet protein, colocalization of these two neuronal markers was revealed in all of type A horizontal cells and alpha ganglion cells and in a small number of AII amacrine cells of the cat retina. Besides the double-labeled neurons, parvalbumin alone was present in type B horizontal cells, in small numbers of starburst- and A13-like amacrine cells and in the somata of unidentified ganglion cells. The processes of the double- or single-labeled amacrine cells did not have a continuous retinal cover. Although the parvalbumin- and neurofilament-immunolabeled amacrine cells belonged to groups of neurons with well-defined cell morphologies, their neurochemical features differed from other AII, starburst and A13 amacrine cells. The presence of these cells may be due to an accidental expression of an unusual combination of neurochemical features during retinal development. It is also possible that these cells support the functioning of ganglion cells with rarely occurring complex receptive fields.