Silvio Varon

Nerve regeneration in silicone chambers: Influence of gap length and of distal stump components

Abstract The range of growth-promoting influences from a distal nerve stump on a regenerating proximal stump was determined using an experimental system in which a gap between cross-anastomosed rat sciatic nerves was encased by a cylindrical silicone chamber. Two arrangements were examined after 1 month in situ: A proximal-distal (PD) system in which both proximal and distal stumps were introduced into the ends of the chamber, and a proximal-open (PO) system in which the distal stump was omitted. When the gap was 6 mm long, a regenerated nerve extended all the way through the chamber in both the PD and PO systems. When the gap was increased to 10 mm, a similar regrowth occurred in the PD chamber, whereas in the PO chamber proximal regrowth was partial or nonexistent. When the gap was increased to 15 mm, no regeneration occurred, even in the presence of the distal stump. These observations confirm that the distal stump influences proximal regeneration and indicate that this influence can act only over a limited distance or volume. Such an influence could consist of humoral agents which support nerve growth and/or outgrowth from the distal stump.

Purification of the Chick Eye Ciliary Neuronotrophic Factor

Abstract: Dissociated 8‐day chick embryo ciliary ganglionic neurons will not survive for even 24 h in culture without the addition of specific supplements. One such supplement is a protein termed the ciliary neuronotrophic factor (CNTF) which is present at very high concentrations within intraocular tissues that contain the same muscle cells innervated by ciliary ganglionic neurons in vivo. We describe here the purification of chick eye CNTF by a 2½‐day procedure involving the processing of intraocular tissue extract sequentially through DE52 ion‐exchange chromatography, membrane ultrafiltration‐concentration, sucrose density gradient ultracentrifugation, and preparative sodium dodecyl sulfate‐polyacrylamide gradient electrophoresis. An aqueous extract of the tissue from 300 eyes will yield about 10–20 μg of biologically active, electrophoretically pure CNTF with a specific activity of 7.5 × 106 trophic units/mg protein. Purified CNTF has an Mr of 20,400 daltons and an isoelectric point of about 5, as determined by analytical gel electrophoresis. In addition to supporting the survival of ciliary ganglion neurons, purified CNTF also supports the 24‐h survival of cultured neurons from certain chick and rodent sensory and sympathetic ganglia. CNTF differs from mouse submaxillary nerve growth factor (NGF) in molecular weight, isoelectric point, inability to be inactivated by antibodies to NGF, ability to support the in vitro survival of the ciliary ganglion neurons, and inability to support that of 8‐day chick embryo dorsal root ganglionic neurons. Thus, CNTF represents the first purified neuronotrophic factor which addresses parasympathetic cholinergic neurons.

Selective survival of neurons from chick embryo sensory ganglionic dissociates utilizing serum-free supplemented medium☆

Dissociated embryonic chick dorsal root ganglionic cells were plated on collagen-coated tissue culture dishes in Eagles basal medium containing 10% fetal calf serum (FCS). After 48 h, which allowed adequate cell attachment, the cultures were washed with serum-free medium and then received fresh medium supplemented with 10% FCS or serum-free defined medium (N1), which was supplemented with insulin, transferrin, progesterone, putrescine and selenium. In addition, both media required the addition of Nerve Growth Factor (NGF). N1 medium selectively maintained the neurons and did not support proliferation or even survival of almost all non-neuronal elements (fibroblasts and Schwann cells). Survival of neurons in N1 was initially as good and eventually better than in serum-containing medium. After 6 days in N1 the cultures consisted almost entirely of neurons (>95%), which had smaller cell bodies but more extensive process formation than in serum-supplemented medium. The omission of any one of the supplements resulted in a reduction of neuron survival. The ability to generate cultures of pure neurons in a serum-free defined medium may be useful for studying (i) the role of specific hormones and growth factors normally supplied by serum in the maintenance of neurons and (ii) biochemical parameters of neurons in the absence of the substantial background due to non-neuronal elements.

Retrograde cell changes in medial septum and diagonal band following fimbria-fornix transection: Quantitative temporal analysis

Complete unilateral fimbria-fornix transections, including the overlying cingulate cortex, were administered to female rats. At time points from 1 day to 6 weeks, the septal-diagonal band region was examined using acetylcholinesterase histochemistry, Cresyl Violet cell staining, and choline acetyltransferase biochemistry. As early as 1 day following the transection a decrease in acetylcholinesterase positive cell body staining was observed in the medial septum; however, no loss of Nissl-stained neurons was measured in Cresyl Violet stained sections until 1 week after the lesion. Maximal loss of acetylcholinesterase-positive cells, as visualized after irreversible acetylcholinesterase inhibition, was measured at 1 week, and no further change was observed at time points up to 6 weeks after operation. The loss of acetyltransferase-positive cells was greatest in the medial septal area (-65%) and the vertical limb of the diagonal band (-55%). Little cell loss was measured in the horizontal limb of the diagonal band. This is consistent with the known projections of these cell bodies. Remaining acetylcholinesterase-positive cell bodies in the medial septum had shrunk by about 20% (measured as the diameter along the major axis). A marked neuronal cell loss (about 50%) was demonstrable in the medial septum and vertical limb of the diagonal band in the Cresyl Violet-stained sections, too. A pile-up of acetylcholinesterase-stained material was observed in the dorsal-lateral quadrant of the septal area just proximal to the lesion at 1 day following transection. This pile-up occurred in the medial septum and diagonal band area up to 1 week following the transection, and had nearly disappeared by 2 weeks post-transection. Choline acetyltransferase biochemical activity, measured in samples of whole septum, decreased significantly at 1 day but subsequently returned to control levels. By 2 weeks following transection, an increase in acetylcholinesterase-positive stained fibers was observed in the dorsal-lateral quadrant of the septum, ipsilateral to the lesion relative to the contralateral septum. This response, which was interpreted as sprouting from the lesioned axons proximal to the transection, probably accounted for the rise in choline acetyltransferase biochemical activity in the whole septum following the reduction on the first day.

Purification of adult rat sciatic nerve ciliary neuronotrophic factor

The ciliary neuronotrophic factor (CNTF), a protein required for the survival of cultured avian embryonic parasympathetic ciliary ganglionic neurons, was recently purified from extracts of selected chick intraocular tissues. Here we report the purification of a mammalian CNTF activity from extracts of adult rat sciatic nerve using a fractionation procedure similar to that employed for isolating chick eye CNTF. About 2 micrograms of CNTF protein can be obtained from each 1.5 g batch of nerve tissue. Like the chick CNTF, the mammalian factor displays trophic activity for dorsal root and sympathetic as well as ciliary ganglionic neurons. The nerve CNTF activity differs from its chick counterpart in molecular weight and chromatographic behavior on ion-exchange columns. Unlike purified nerve growth factor (NGF), nerve CNTF activity is insensitive to anti-NGF antibodies and is unable to support the survival of 8-day chick embryo dorsal root ganglion neurons.

Delayed treatment with nerve growth factor reverses the apparent loss of cholinergic neurons after acute brain damage

Previous studies have shown that the loss after brain injury of adult rat septal cholinergic neurons whose axons are transected can be prevented by immediate intraventricular nerve growth factor (NGF) administration. This loss of axotomized neurons may be due to a reduction in detectability of neurotransmitter-related enzyme rather than to neuronal death. Here we report that NGF treatment, started after most of the neurons were no longer detectable (i.e., 1, 2, and 3 weeks), induced a dramatic reappearance of the apparently lost cholinergic neurons. These results may have important implications for potential trophic factor treatments of CNS trauma and neurodegenerative diseases, such as Alzheimers dementia, which are characterized by chronic and progressive losses in the function of specific sets of neurons.

Nerve regeneration model and trophic factors in vivo

The proximal stump of a transected rat sciatic nerve has been observed to regenerate through a cylindrical silicone chamber across a 10 mm gap to the distal stump. The fluid filling such in vivo chambers contains trophic factors that ensure in vitro survival and growth of at least sensory neurons from rodent dorsal root ganglia--as already demonstrated for fluid generated in vitro from Schwann and other cell cultures.

An automated colorimetric microassay for neuronotrophic factors

A microassay is described for determining the number of neurons surviving after 24 h in response to added neuronotrophic factors. Neuronal cultures in 96-well microtiter plates are supplied with a yellow tetrazolium derivative, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), which is taken up selectively by viable neurons and converted to a blue formazan product. The amount of blue color development can be rapidly quantified using an automatic microplate spectrophotometer. The resulting optical density is directly proportional to the number of viable neurons. The spectrophotometer has been interfaced with a computer allowing a print out of individual absorbance values and calculation of half-maximal (one trophic unit) neuronal survival. The assay has been used for the quantification of the trophic activities of nerve growth factor and ciliary neuronotrophic factor using, respectively, dorsal root and ciliary ganglionic neurons from 8-day chick embryos. Assay parameters were optimized so that about 2000 individual cultures of ganglionic neurons can be set up and analyzed each day, thus allowing the serial titration in duplicate of 80-120 separate samples. The determination of neuronal number and titer calculation steps now requires about 2 min per microplate (96 cultures), a 50-fold reduction in time over existing methods.

Neurite-promoting factors and extracellular matrix components accumulating in vivo within nerve regeneration chambers

The outgrowth of neurites from cultured neurons can be induced by the extracellular matrix glycoproteins, fibronectin and laminin, and by polyornithine-binding neurite-promoting factors (NPFs) derived from culture media conditioned by Schwann, or other cultured cells. We have examined the occurrence of fibronectin, laminin and NPFs during peripheral nerve regeneration in vivo. A previously established model of peripheral nerve regeneration was used in which a transected rat sciatic nerve regenerates through a silicone chamber bridging a 10 mm interstump gap. The distribution of fibronectin and laminin during regeneration was assessed by indirect immunofluorescence. Seven days after nerve transection the regenerating structure within the chamber consisted primarily of a fibrous matrix which stained with anti-fibronectin but not anti-laminin. At 14 days, cellular outgrowths from the proximal and distal stumps (along which neurites grow) had entered the fibronectin-containing matrix, consistent with a role of fibronectin in promoting cell migration. Within these outgrowths non-vascular as well as vascular cells stained with anti-fibronectin and anti-laminin. Within the degenerated distal nerve segment, cell characteristic of Bungner bands (rows of Schwann cells along which regenerating neurites extend) stained with anti-fibronectin and laminin. The fluid surrounding the regenerating nerve was found to contain NPF activity for cultured ciliary ganglia neurons which markedly increased during the period of neurite growth into the chamber. In previous studies using this particular neurite-promoting assay, laminin but to a much lesser extent fibronectin also promoted neurite outgrowth.(ABSTRACT TRUNCATED AT 250 WORDS)

Ciliary neurotrophic factor prevents neuronal degeneration and promotes low affinity NGF receptor expression in the adult rat CNS

Recombinant human ciliary neurotrophic factor (CNTF) was infused for 2 weeks into the lateral ventricle of fimbria-fornix transected adult rats, and its effects were compared with those of purified mouse nerve growth factor (NGF). We provide evidence that CNTF can prevent degeneration and atrophy of almost all injured medial septum neurons (whereas NGF protects only the cholinergic ones). CNTF is also involved in up-regulation of immunostainable low affinity NGF receptor (LNGFR) in cholinergic medial septum and neostriatal neurons and in a population of lateral septum neurons. In contrast to NGF, CNTF did not stimulate choline acetyltransferase in the lesioned septum and normal neostriatum (pointing to different mechanisms for the regulation of choline acetyltransferase and LNGFR), cause hypertrophy of septal or neostriatal cholinergic neurons, or cause sprouting of LNGFR-positive (cholinergic) septal fibers.