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Biomedical subjects

A Bignami

Publications and source records attributed to A Bignami.

At least 37 records · Page 2Linked to original sources
PubMed1991

Neurofilament phosphorylation in the axonless horizontal cells of rat retina.

Axonless horizontal cells in the outer plexiform layer of rat retina were studied with 19 monoclonal antibodies reacting with phosphorylated and non-phosphorylated epitopes of the two high molecular weight neurofilament proteins (NF 150K and NF 200K). With 6 antibodies, immunoreactivity was confined to the nerve fiber layer on the inner surface of the retina. Horizontal cells were not stained. Four antibodies in this group were axon-specific, while the remaining two stained motor and sensory neuron perikarya in rat spinal cord and dorsal root ganglia, respectively. Of the 13 antibodies which stained horizontal cells, 11 reacted with phosphorylated epitopes and failed to decorate motor neuron perikarya in the spinal cord, while in dorsal root ganglia, they stained a subpopulation of sensory neurons.

Animals↗
PubMed1991

Extracellular matrix of central nervous system white matter: demonstration of an hyaluronate-protein complex.

Monoclonal antibodies were raised against human glial hyaluronate-binding protein (GHAP), a major CNS-specific glycoprotein known to bind hyaluronate in vitro. Frozen sections of dog and human spinal cord were digested with Streptomyces hyaluronidase in order to ascertain whether GHAP is bound to hyaluronate in vivo. Digestion with hyaluronidase, prior to staining of the sections by conventional indirect immunofluorescence, led to a drastic reduction in the intensity of the staining reaction. Chondroitinase ABC (protease-free) was also effective in bringing about the release of GHAP from tissue sections. This enzyme also degrades hyaluronate. The effects of the chondroitinase were completely reversed by the addition of 1 mM Zn2+, a known inhibitor of this enzyme. The intact protein was released into the soluble fraction of human brain homogenates by testicular hyaluronidase. An immunoreactive species of 70 kD was released into the soluble fraction of dog spinal cord homogenates by Streptomyces hyaluronidase. Dog GHAP was isolated from spinal cord by means of ion exchange and affinity chromatography. This protein bound efficiently to hyaluronate in vitro. Dog and human GHAP had identical isoelectric points and similar peptide maps but different molecular weights. Dog GHAP (70 kD) was larger than its human counterpart (60 kD). These findings imply that GHAP exists in association with hyaluronate in CNS white matter. Immunoelectron microscopy revealed that GHAP fills the space between myelin sheaths in dog spinal cord white matter. One is led to conclude therefore that an hyaluronate based extracellular matrix exists in CNS white matter.

Amidohydrolases↗
PubMed1991

Localization of hyaluronate in primary glial cell cultures derived from newborn rat brain.

We have devised a technique that enables one to localize hyaluronate in cultured cells. Cells were probed with the glial hyaluronate binding protein (GHAP) which was itself then visualized by conventional indirect immunofluorescence. The hyaluronate binding properties of this protein have been established. This technique was applied to the study of hyaluronate synthesis in glial cells. These cells do not themselves produce GHAP. O-2A progenitor cells were obtained from the cerebral hemispheres of newborn rats. These cells are bipotential in that they are able to differentiate into either oligodendrocytes or type 2 astrocytes depending on the composition of the culture medium. In cultures of O-2A progenitor cells maintained in the absence of serum, in which large numbers of oligodendrocytes appeared, very little hyaluronate was produced. The galC+ cells were invariably hyaluronate negative. Cultures of the same cells, maintained in the presence of 10% FCS, contained large numbers of hyaluronate producing cells. The hyaluronate producing cells were typically small, process-bearing, and GFAP+. Some, but not all, were A2B5+ and could, therefore, be identified as type 2 (GFAP+, A2B5+) astrocytes. Type 1 (GFAP+, A2B5-) astrocytes were also active in the synthesis of hyaluronate, to the extent that they were able to coat their substrate with hyaluronate. Among cells of the O-2A lineage, then, hyaluronate production would appear to be restricted to astrocytes. This may have some bearing on the origin of hyaluronate in the extracellular matrix of CNS white matter.

Animals↗
PubMed1991

Astrocytes colonize dorsal root ganglia transplanted into rat brain.

Fragments of dorsal root ganglia (DRG) were grafted into rat brain and examined one month later. The autografts were similar to their normal counterparts when stained with toluidine blue or by indirect immunofluorescence with laminin and neurofilament antibodies. However, a major difference was observed with antibodies to the glial fibrillary acidic protein (GFAP). Normal DRG were GFAP-negative while the autografts were intensely and diffusely stained. The GFAP antibodies used in this study did not decorate Schwann cells or satellite cells in peripheral nerve and DRG, and thus appeared to recognize the "central" form of GFAP (17). Thus reactive astrocytes appear to be capable of migration into grafted nervous tissues without producing apparent neuronal damage.

Animals↗
PubMed1990

Glial hyaluronate-binding protein (GHAP) in optic nerve and retina.

The distribution of glial fibrillary acidic protein (GFAP) and of glial hyaluronate-binding protein (GHAP) was studied by indirect immunofluorescence with monoclonal and polyclonal antibodies in dog, rat and rabbit optic nerve. In dog and rabbit, myelination extends into the optic nerve head inside the eye, while in the rat myelination of the optic nerve ceases abruptly at its entry into the eye. Outside the eye the distribution of the two proteins was similar. Both antigens formed a delicate mesh surrounding myelinated optic nerve axons. In all 3 species GFAP immunoreactivity continued uninterrupted into the optic nerve head inside the eye. Conversely, in both dog and rat, GHAP immunoreactivity ceased abruptly in the region of the lamina cribrosa, a sieve-like structure continuous with the sclera through which bundles of optic nerve axons pass. No staining was observed in the myelinated optic nerve head of the dog nor in the non-myelinated optic nerve head of the rat. In the rabbit lacking a lamina cribrosa, GHAP immunoreactivity did not cease abruptly at the optic nerve entry into the eye, but the staining intensity was reduced in the optic nerve head.

Animals↗
PubMed1990

Permissive and non-permissive reactive astrocytes: immunofluorescence study with antibodies to the glial hyaluronate-binding protein.

Two distinct types of reactive astrocytes were studied in rat CNS. Reactive astrocytes secondary to penetrating trauma (anisomorphic gliosis) were induced by stab wounds to the brain. Reactive astrocytes secondary to Wallerian degeneration (isomorphic gliosis) were induced in spinal cord dorsal columns by dorsal rhizotomy proximal to dorsal root ganglia. Anisomorphic glial scars did not stain with antibodies to the glial hyaluronate-binding protein (GHAP), a structural glycoprotein of white matter extracellular matrix. Conversely, isomorphic glial scars were still GHAP-positive 3 months after dorsal root transection. Only after 5 months did GHAP immunoreactivity start to disappear from the isomorphic glial scar. Extensive dorsal rhizotomy was performed at the lumbar level to produce Wallerian degeneration of spinal cord dorsal columns. One month later, the rats were reoperated and two thoracic dorsal roots were implanted in the degenerated dorsal columns. The rats were examined 1 month after grafting. As expected, there was a dense anisomorphic glial scar at the site of surgery, while the dorsal columns above the graft showed isomorphic gliosis. Extensive axonal growth was observed in the dense glial scar surrounding the graft. Conversely, no axonal growth was observed in the degenerated dorsal columns undergoing isomorphic gliosis above the implant. The findings suggested that GHAP-negative astrocytes responding to traumatic injury are permissive for axonal growth and that GHAP-positive astrocytes responding to Wallerian degeneration are not permissive.

Animals↗
PubMed1989

Neurofilament phosphorylation in neuronal perikarya following axotomy: a study of rat spinal cord with ventral and dorsal root transection.

Rat spinal cord was stained by indirect immunofluorescence with 11 neurofilament monoclonal antibodies that recognize phosphorylated epitopes. All monoclonals were axon-specific in this location. The large motoneurons containing bundles of neurofilaments did not stain and the pattern remained unchanged after transection of the sciatic nerve in the thigh. With nine monoclonals, stained motoneurons were observed in the ventral horns 3 days, 5 days, 1 week, and 2 weeks after transection of the ventral roots close to the spinal cord. The abnormal motoneurons were typically scattered among normal (i.e., nonstained) cells. Even in animals showing the most severe reaction, the whole motoneuron population at the site of rhizotomy was not affected, stained and nonstained perikarya often coexisting side by side. Stained motoneurons were no longer observed 3 weeks after ventral root transection. Changes in neuronal immunoreactivity were also observed after dorsal root transection. However, a different population was affected, i.e., middle-sized neurons in dorsal horns and at the base of ventral horns. With two monoclonals (A9 and D21), cell bodies remained negative following all operations. It is concluded that axotomy in proximity of the cell body may induce certain neurofilament phosphorylation events in motor neuron perikarya, whereas other phosphorylation events remain confined to the axons under these experimental conditions. The absence of changes after transection of the sciatic nerve in the thigh suggests that neurofilament phosphorylation is a reaction to cell injury rather than a cellular event related to nerve regeneration.

Animals↗
PubMed1989

Isolation and partial characterization of a glial hyaluronate-binding protein.

A glial hyaluronate-binding protein (GHAP) with an isoelectric point of 4.3-4.4 was isolated from human brain white matter. The 60-kDa glycoprotein appeared to be quite resistant to proteolysis, and comparison with GHAP from a viable glioma removed at surgery showed that the protein isolated from autopsy material was not a degradation product resulting from postmortem autolysis. The protein was localized immunohistochemically with mouse monoclonal and rabbit polyclonal antibodies in cerebral white matter. Only small amounts could be found in the gray matter. After enzymatic deglycosylation, an immunoreactive 47-kDa polypeptide was obtained. Two amino acid sequences of GHAP showed a striking similarity (up to 89%) with a highly conserved region of cartilage proteins (bovine nasal cartilage proteoglycan and rat and chicken link protein). However, the amino acid composition and other amino acid sequences suggested that there are also differences between brain-specific GHAP and cartilage proteins.

Amino Acid Sequence↗
PubMed1989

Glial hyaluronate-binding protein in Wallerian degeneration of dog spinal cord.

Wallerian degeneration of spinal cord dorsal columns was produced in three dogs by unilateral extradural dorsal rhizotomy at the lower thoracic level. The spinal cord was studied 1 month, 2 months, and 3 months after surgery. Transverse cryostat sections at the site rhizotomy and at the mid-thoracic level were stained by indirect immunofluorescence with antibodies to the glial fibrillary acidic protein (GFAP) and to the glial hyaluronate-binding protein (GHAP). GHAP immunoreactivity was almost unchanged in the degenerated dorsal column 1 month after rhizotomy. After 2 and 3 months, staining with GHAP antibodies was markedly decreased in the gliosed dorsal column at the site of rhizotomy, but it still persisted at the mid-thoracic level. It is concluded that GHAP persists for long periods of time in dorsal columns undergoing Wallerian degeneration, a finding consistent with its putative role as a nonpermissive substrate preventing nerve regeneration in CNS white matter.

Animals↗
PubMed1989

Axonal regeneration in old multiple sclerosis plaques. Immunohistochemical study with monoclonal antibodies to phosphorylated and non-phosphorylated neurofilament proteins.

Cryostat sections of two old plaques removed at autopsy from the spinal cord of a 62-year-old man with multiple sclerosis of 24-year duration were studied by indirect immunofluorescence with antibodies to neurofilament proteins, glial fibrillary acidic protein (GFAP), glial hyaluronate-binding protein (GHAP), vimentin and laminin. The neurofilament monoclonal antibodies used in this study reacted with phosphorylated epitopes of the two large polypeptides of the neurofilament triplet (NF 150K, NF 200K). As previously reported [Dahl D, Labkovsky B, Bignami A (1989) Brain Res Bull 22:225-232], the neurofilament antibodies either stained axons in the distal stump of transected sciatic nerve in the early stages of regeneration or late in the process, i.e., after regenerating axons had reached the distal stump of the transected sciatic nerve. Both multiple sclerosis plaques were positive for GFAP and vimentin, but negative for GHAP, while astrocytes in myelinated spinal cord white matter stained with both GFAP and GHAP antibodies. Laminin immunoreactivity in the plaques and normal spinal cord was confined to blood vessels. One plaque was almost devoid of axons as evidenced by indirect immunofluorescence with neurofilament antibodies. Another plaque was packed with bundles of thin axons running an irregular course in the densely gliosed tissue. Axons in the plaque only stained with neurofilament antibodies reacting with sciatic nerve in the early stages of regeneration while axons in the surrounding myelinated white matter were decorated by all neurofilament antibodies, regardless of the time of appearance of immunoreactivity in crushed sciatic nerve. It is concluded that reactive astrocytes forming glial scars do not constitute a non-permissible substrate for axonal growth.

Antibodies, Monoclonal↗
PubMed1989

Glial hyaluronate-binding protein in dysmyelinating mice mutants: jimpy, quaking and shiverer.

Cryostat sections of cerebral hemispheres, cerebellum and spinal cord from dysmyelinating mice mutants (quaking, jimpy and shiverer) and littermate controls were stained by indirect immunofluorescence with polyclonal antibodies to the glial hyaluronate-binding protein (GHAP), a brain-specific extracellular matrix glycoprotein produced by astrocytes. In normal mice, the distribution of GHAP was similar to that previously reported in human, calf, pig and dog. The antigen was mainly localized in white matter, the granular layer of the cerebellum being the main exception. No differences were observed between mutants and littermate controls, except that with both GHAP and glial fibrillary acidic protein antibodies the glial framework was denser in the mutants, probably due to the reduction in myelin. The findings suggest that GHAP expression by astrocytes is not induced by myelination and that white matter astrocytes constitute a distinct glial population.

Animals↗
PubMed1989

Intermediate filament proteins immunologically related to desmin in astrocytes: a study of chicken spinal cord by two-dimensional gel electrophoresis and immunoblotting.

Co-migration experiments by two-dimensional SDS-PAGE using chicken spinal cord extracts and desmin purified from chicken gizzard showed that desmin is not present in spinal cord. However, by the immunoblotting procedure, desmin antibodies recognized 3 spinal cord antigens with different molecular weights and isoelectric points than desmin and the glial fibrillary acidic (GFA) protein. These antigens which also reacted with GFA protein antibodies were not identified in chicken gizzard extracts. The reactivity of the antigens with a monoclonal antibody recognizing an epitope common to most intermediate filament proteins (1) suggests that immunostaining of astrocytes with desmin antibodies (2, 3) is due to the presence of new intermediate filament proteins immunologically related to desmin.

Animals↗
PubMed1989

Early and late appearance of neurofilament phosphorylation events in nerve regeneration.

Neurofilament phosphorylation in regenerating rat sciatic nerve was studied by indirect immunofluorescence with monoclonal antibodies reacting with phosphorylated epitopes of the 2 large polypeptides of the neurofilament protein triplet (NF 150K, NF 200K). One group of antibodies decorated axons early in the process. In fact, no differences were seen in double labeled sections between these antibodies and polyclonal neurofilament antibodies as to their reactivity with the distal stump of transected sciatic nerves. Another group stained axons after they had completed their elongation, i.e., after they had reached the distal part of the denervated sciatic nerve. In general, the epitopes recognized by antibodies in this group appeared more sensitive to phosphatase digestion as compared to the first group. Furthermore, there was a good correlation between the thickness of the regenerated axons and staining with these monoclonal antibodies. Thick axons (like those observed in normal nerves) were stained, while bundles of thin axons remained unstained. Monoclonal II32 stained regenerated axons in a remarkable segmental pattern. With this antibody, continuous decoration of the axons was still not observed 7 weeks after transection, the longest follow-up period in this study. We suggest that some neurofilament phosphorylation events may contribute to the stabilization of the axonal cytoskeleton and that abnormalities persist in regenerated axons as to the extent of neurofilament phosphorylation.

Animals↗
PubMed1989

Structural similarity of hyaluronate binding proteins in brain and cartilage.

A glial hyaluronate-binding protein (GHAP) was isolated from human brain white matter by affinity chromatography on immobilized hyaluronate. The 60 kDa protein appeared remarkably homogeneous by reversed-phase high pressure liquid chromatography analysis. Four cyanogen bromide peptides and 10 tryptic peptides were characterized by amino acid sequence, a total of 12 sequences since overlaps were found between 2 cyanogen bromide and 2 tryptic peptide sequences. Two sequences of brain GHAP had similarity with rat link protein, a hyaluronate binding protein in cartilage. The region of similarity was contained in the evolutionary conserved COOH-terminal half of link protein which is involved in the binding of hyaluronate. The remaining 10 amino acid sequences of brain GHAP had no similarity with link protein, nor with previously reported protein sequences. The findings suggest that the hyaluronate binding domains of such diverse proteins as brain GHAP and cartilage link protein are similar, probably due to the fact that hyaluronic acid is highly conserved in evolution.

Amino Acid Sequence↗
PubMed1989

Vimentin-GFAP transition in primary dissociated cultures of rat embryo spinal cord.

Primary dissociated cultures derived from 15-day-old rat embryo spinal cord with or without dorsal root ganglia (DRG) were grown on polylysine, Primaria and laminin substrates. On polylysine and Primaria substrates, spinal cord neurons formed aggregates connected by bundles of neurites in a distinctive pattern similar to that observed in cultures derived from embryonal rat brain and neonatal rat cerebellum. After 2 days in culture, the number of cells stained with GFAP antibodies progressively increased within the vimentin-positive monolayer surrounding the neuronal aggregates. These astrocytes had the typical appearance of astrocytes in primary dissociated cultures derived from late fetal or early neonatal murine brain, i.e. large flat or stellate cells with thick processes staining equally well with GFAP and vimentin antibodies. Astrocytes found within the neuronal aggregates in 4-5 day cultures were markedly different, i.e. small stellate cells with slender processes forming a delicate mesh throughout the aggregate. These GFAP-positive cells stained only weakly with vimentin antibodies. Spinal cord neurons formed aggregates on laminin substrates but failed to extend neurites and rapidly degenerated. The large flat cells in the surrounding monolayer gradually invaded the aggregates. These cells stained with both GFAP and vimentin antibodies. DRG neurons developed equally well on Primaria and laminin substrates, extending their neurites on the vimentin-positive flat cells forming the monolayer regardless of their reactivity with GFAP antibodies.

Animals↗
PubMed1989

A case of post-traumatic syringomyelia. Neuropathological findings after 1 year of cystic drainage.

A 58-year-old man sustained C4-C5 post-traumatic myelopathy with C3-C4 subluxation, slight compression of C3 vertebral body, C4 spinous process fracture and C6 compression fracture. He subsequently developed syringomyelia from C4 to C6, which was shunted into the subarachnoid space. Postoperatively, there was some but insignificant improvement of his symptoms although a postoperative metrizamide spinal computerised tomography showed complete drainage of the cyst. This patient died 1 year later. Autopsy examination of the spinal cord showed extensive damage of the posterior half of the cord at C3-C4 but the damage was much less extensive from C4 to C6 (where the syrinx was located), affecting mainly the right dorsal column at C4 and the right dorsal column and right anterior horn at C5-C6. In this patient, the syrinx developed in the partially damaged segments of the cord at the level of the spinal fractures and complete drainage of the cyst was not followed by satisfactory relief of his symptoms.

Humans↗