Incorporation and glial differentiation of mouse EGF-responsive neural progenitor cells after transplantation into the embryonic rat brain.

In vitro, epidermal growth factor (EGF)-responsive neural progenitor cells exhibit multipotent properties and can differentiate into both neurons and glia. Using an in utero xenotransplantation approach we examined the developmental potential of EGF-responsive cells derived from E14 mouse ganglionic eminences, cortical primordium, and ventral mesencephalon, after injection into the E15 rat forebrain ventricle. Cell cultures were established from control mice or from mice carrying the lacZ transgene under control of the promoters for nestin, glial fibrillary acidic protein (GFAP), or myelin basic protein (MBP). The grafted cells, visualized with mouse-specific markers or staining for the reporter gene product, displayed widespread incorporation into distinct forebrain and midbrain structures and differentiated predominantly into glial cells. The patterns of incorporation of cells from all three regions were very similar without preference for the homotopic brain areas. These results suggest that EGF-responsive progenitor cells can respond to host derived environmental cues, differentiate into cells with glial-like features, and become integrated in the developing recipient brain.

Generation and transplantation of EGF-responsive neural stem cells derived from GFAP-hNGF transgenic mice.

EGF-responsive neural stem cells isolated from murine striatum have the capacity to differentiate into both neurons and glia in vitro. Genetic modification of these cells is hindered by a number of problems such as gene stability and transfection efficiency. To circumvent these problems we generated transgenic mice in which the human GFAP promoter directs the expression of human NGF. Neural stem cells isolated from the forebrain of these transgenic animals proliferate and form clusters, which appear identical to stem cells generated from control animals. Upon differentiation in vitro, the transgenic stem cell-derived astrocytes express and secrete bioactive hNGF. Undifferentiated GFAP-hNGF or control stem cells were transplanted into the striatum of adult rats. One and 3 weeks after transplantation, hNGF was detected immunocytochemically in an halo around the transplant sites. In GFAP-hNGF-grafted animals, intrinsic striatal neurons proximal to the graft appear to have taken up hNGF secreted by the grafted cells. Ipsilateral to implants of GFAP-hNGF-secreting cells, choline acetyltransferase-immunoreactive neurons within the striatum were hypertrophied relative to the contralateral side or control-grafted animals. Further, GFAP-hNGF-grafted rats displayed a robust sprouting of p75 neurotrophin receptor-positive fibers emanating from the underlying basal forebrain. These studies indicate that EGF-responsive stem cells which secrete hNGF under the direction of the GFAP promoter display in vitro and in vivo properties similar to that seen following other methods of NGF delivery and this source of cells may provide an excellent avenue for delivery of neurotrophins such as NGF to the central nervous system.

Grafts of EGF-responsive neural stem cells derived from GFAP-hNGF transgenic mice: trophic and tropic effects in a rodent model of Huntington's disease.

The present study examined whether implants of epidermal growth factor (EGF)-responsive stems cells derived from transgenic mice in which the glial fibrillary acid protein (GFAP) promoter directs the expression of human nerve growth factor (hNGF) could prevent the degeneration of striatal neurons in a rodent model of Huntington's disease (HD). Rats received intrastriatal transplants of GFAP-hNGF stem cells or control stem cells followed 9 days later by an intrastriatal injection of quinolinic acid (QA). Nissl stains revealed large striatal lesions in rats receiving control grafts, which, on average, encompassed 12.78 mm3. The size of the lesion was significantly reduced (1.92 mm3) in rats receiving lesions and GFAP-hNGF transplants. Rats receiving QA lesions and GFAP-hNGF-secreting grafts stem cell grafts displayed a sparing of striatal neurons immunoreactive (ir) for glutamic acid decarboxylase, choline acetyltransferase, and neurons histochemically positive for nicotinamide adenosine diphosphate. Intrastriatal GFAP-hNGF-secreting implants also induced a robust sprouting of cholinergic fibers from subjacent basal forebrain neurons. The lesioned striatum in control-grafted animals displayed numerous p75 neurotrophin-ir (p75NTR) astrocytes, which enveloped host vasculature. In rats receiving GFAP-hNGF-secreting stem cell grafts, the astroglial staining pattern was absent. By using a mouse-specific probe, stem cells were identified in all animals. These data indicate that cellular delivery of hNGF by genetic modification of stem cells can prevent the degeneration of vulnerable striatal neural populations, including those destined to die in a rodent model of HD, and supports the emerging concept that this technology may be a valuable therapeutic strategy for patients suffering from this disease.

Myelination following transplantation of EGF-responsive neural stem cells into a myelin-deficient environment.

Epidermal growth factor (EGF)-responsive stem cells have been identified in the murine central nervous system. These cells can be isolated from the brain and maintained in an undifferentiated state in vitro in the presence of EGF. After removing EGF, the cells cease mitosis and can be induced to differentiate into neurons, astrocytes, and oligodendrocytes. We demonstrate that when the undifferentiated stem cells (nestin-positive) are injected into the myelin-deficient rat spinal cord, they respond to cues within the mutant CNS and differentiate into myelinating oligodendrocytes, in contrast to their behavior in vitro, where they mainly form astrocytes. The cells provide a valuable model system for the study of the development of early oligodendrocytes from multipotent neural stem cells. Because these cells are influenced to divide using growth factors, rather than oncogenes, and because they appear to make appropriate lineage decisions when transplanted into a mutant environment, they may provide an excellent source of cells for a variety of future therapies using cellular transplantation.

Cografting with polymer-encapsulated human nerve growth factor-secreting cells and chromaffin cell survival and behavioral recovery in hemiparkinsonian rats.

Encapsulated cell grafting is one approach for the delivery of neurotransmitters and/or neurotrophic factors to the brain. Baby hamster kidney (BHK) cells were genetically modified to secrete high levels of human nerve growth factor (hNGF). Following polymer encapsulation, these cells were implanted into the left lateral ventricle or the left striatum 1.5 mm away from striatally cografted unencapsulated adrenal medullary chromaffin cells in hemiparkinsonian rats. Although the animals receiving adrenal medulla alone or adrenal medulla with intraventricular hNGF-secreting cell grafting did not show recovery of apomorphine-induced rotational behavior, the animals receiving adrenal medulla with intrastriatal hNGF-secreting cell implants showed a significant recovery of rotational behavior 2 and 4 weeks after transplantation. Histological analysis revealed that in animals receiving adrenal medulla with intraventricular hNGF-secreting cell grafting, the number of tyrosine hydroxylase-immunoreactive (TH-IR) surviving chromaffin cells tended to be higher (approximately five to six times) than in animals receiving adrenal medulla alone; however, this increase did not reach statistical significance. In contrast, in animals receiving adrenal medullary cells together with intrastriatal hNGF-secreting cells, the number of TH-IR surviving chromaffin cells was more than 20 times higher than that in animals receiving adrenal medullary cells alone. Analysis of retrieved capsules revealed that hNGF continued to be released by encapsulated BHK-hNGF cells after 4 weeks in vivo. Moreover, histological analysis confirmed the presence of numerous viable encapsulated BHK-hNGF cells. These results indicate the potential use of intrastriatal implantation of encapsulated hNGF-secreting cells for augmenting the survival of cografted chromaffin cells as well as promoting the functional recovery of hemiparkinsonian rats. These data indicate that this approach may have potential application for treating Parkinson's disease.

Intrathecal delivery of CNTF using encapsulated genetically modified xenogeneic cells in amyotrophic lateral sclerosis patients.

Neuronal growth factors hold promise for providing therapeutic benefits in various neurological disorders. As a means of ensuring adequate central nervous system delivery of growth factors and minimizing significant adverse side effects associated with systemic delivery methods, we have developed an ex vivo gene therapy approach for protein delivery using encapsulated genetically modified xenogeneic cells. Ciliary neurotrophic factor (CNTF) has been shown in various rodent models to reduce the motor neuron cell death similar to that seen in amyotrophic lateral sclerosis (ALS). The initial trials focusing on the systemic administration of CNTF for ALS have been discontinued as a result of major side effects, thus preventing determination of the potential efficacy of the molecule. In order to deliver CNTF directly to the nervous system, we conducted a phase I study in which six ALS patients were implanted with polymer capsules containing genetically engineered baby hamster kidney cells releasing approximately 0.5 microgram of human CNTF per day in vitro. The CNTF-releasing implants were surgically placed within the lumbar intrathecal space. Nanogram levels of CNTF were measured within the patients' cerebrospinal fluid (CSF) for at least 17 weeks post-transplantation, whereas it was undetectable before implantation. Intrathecal delivery of CNTF was not associated with the limiting side effects observed with systemic delivery. These results demonstrate that neurotrophic factors can be continuously delivered within the CSF of humans by an ex vivo gene therapy approach, opening new avenues for the treatment of neurological diseases.

Gene therapy for amyotrophic lateral sclerosis (ALS) using a polymer encapsulated xenogenic cell line engineered to secrete hCNTF.

The gene therapy approach presented in this protocol employs a polymer encapsulated, xenogenic, transfected cell line to release human ciliary neurotrophic factor (hCNTF) for the treatment of Amyotrophic Lateral Sclerosis (ALS). A tethered device, containing around 10(6) genetically modified cells surrounded by a semipermeable membrane, is implanted intrathecally; it provides for slow continuous release of hCNTF at a rate of 0.25 to 1.0 micrograms/24 hours. The semipermeable membrane prevents immunologic rejection of the cells and interposes a physical, virally impermeable barrier between cells and host. Moreover, the device and the cells it contains may be retrieved in the event of side effects. A vector containing the human CNTF gene was transfected into a line of baby hamster kidney cells (BHK) with calcium phosphate using a dihydrofolate reductase-based selection vector with a SV40 promoter and contains a HSV-tk killer gene. hCNTF is a potent neurotrophic factor which may have utility for the treatment of ALS. Systemic delivery of hCNTF in humans has been frustrated by peripheral side effects, the molecule's short half life, and its inability to cross the blood-brain barrier. The gene therapy approach described in this protocol is expected to mitigate such difficulties by local intrathecal delivery of a known quantity of continuously-synthesized hCNTF from a retrievable implant.

GDNF slows loss of motoneurons but not axonal degeneration or premature death of pmn/pmn mice.

Glial cell line-derived neurotrophic factor (GDNF), a member of the TG F-beta superfamily, has been shown to be a highly potent neurotrophic factor that enhances survival of various neuronal cell types including motoneurons. To assess its therapeutic potential in treating neurodegenerative diseases such as amyotrophic lateral sclerosis, we treated mutant mice displaying motoneuron degeneration (progressive motor neuropathy; pmn) with encapsulated GDNF-secreting cells. Effects of GDNF treatment on pmn/pmn mice were compared with previous results obtained with ciliary neurotrophic factor (CNTF) [Sagot Y, Tan SA, Baetge E, Schmalbruch H, Kato AC, Aebischer P (1995) Eur J Neurosci 7:1313-1322]. In contrast to CNTF, GDNF did not increase the lifespan of pmn/pmn mice. However, GDNF significantly reduced the loss of facial motoneurons by 50%, a value similar to what was observed when CNTF was administered to the pmn/pmn mice. Surprisingly, myelinated axon counts revealed that GDNF had no effect on nerve degeneration. Therefore, despite its potential in rescuing motoneuron cell bodies, the inability of GDNF to prevent nerve degeneration in pmn/pmn mice suggests that its usefulness in the treatment of motor neuron diseases may be restricted to cotreatment with other factors that act on the nerve process.

Mutagenesis of ser41 to ala inhibits the association of GAP-43 with the membrane skeleton of GAP-43-deficient PC12B cells: effects on cell adhesion and the composition of neurite cytoskeleton and membrane.

To investigate the molecular basis for GAP-43 function in axon outgrowth, we produced a mutant, GAP-43 (Ala41), whose interaction with calmodulin in vitro was unaffected by increasing Ca2+ concentrations, and stably transfected it into GAP-43-deficient PC12B cells. Several lines that expressed wild-type or mutant protein at levels that resembled endogenous GAP-43 expression in PC12 controls were subcloned and characterized. GAP-43 (Ala41) was significantly more extractable with Nonidet P-40 and less tightly associated with the membrane skeleton than the wild-type protein. Furthermore, GAP-43 (Ala41) expression by PC12B cells profoundly affected their phenotype: First, observation of living cells using video-enhanced microscopy revealed irregular plasma membranes with numerous blebs and protrusions and neurites that appeared thin and varicose. Second, both the cells' ability to remain attached to laminin substrates and the amount of alpha 1 beta 1 integrin expressed on the cell surface was significantly decreased. Finally, peripherin transport, which is abnormal in PC12B cells, could be rescued by transfection of wild-type GAP-43 but not the GAP-43 (Ala41) mutant. The phenotypic abnormalities resemble other cell types in which membrane skeleton/plasma membrane interactions have been functionally decoupled, and our results are consistent with the notion that these interactions may be abnormal in GAP-43 (Ala41)-expressing PC12B cells, either as a direct consequence of the mutation or arising secondarily to the altered availability of calmodulin in the growing neurite.

Reduced electrical excitability of PC12 cells deficient in GAP-43: comparison with GAP-43-positive cells.

The electrical excitability of 3 lines of rat pheochromocytoma (PC12) cells were determined under current-clamp recording conditions. In the presence of nerve growth factor (NGF), PC12(A) 'control' cells expressed high levels of GAP-43 protein, PC12(B) cells were highly deficient in GAP-43, and PC12(AB) cells, created by transfection of PC12(B) cells with a rat GAP-43 gene construct, expressed high levels of GAP-43. All 3 lines had similar resting membrane potentials, but significantly greater proportions of GAP-43-containing PC12(A) and PC12(AB) cells exhibited spiking in response to depolarizing current pulses. These spikes were resistant to TTX, were greatly enhanced in TEA and TTX, and were substantially reduced by L-type Ca(2+)-channel antagonists. GAP-43 expression may regulate PC12 cell excitability following NGF treatment, as reflected in a lower proportion of cells capable of discharging with Ca(2+)-spikes in a GAP-43-deficient cell line.

Implantation of encapsulated catecholamine and GDNF-producing cells in rats with unilateral dopamine depletions and parkinsonian symptoms.

Studies in rodents suggest that PC12 cells, encapsulated in semipermeable ultrafiltration membranes and implanted in the striatum, have some potential efficacy for the treatment of age- and 6-OHD-induced sensorimotor impairments (22, 70, 71, 74). The objectives of this study were to: (1) determine if baby hamster kidney cells engineered to secrete glial cell line-derived neurotrophic factor (BHK-GDNF) would survive encapsulation and implantation in a dopamine-depleted rodent striatum, (2) compare polymer-encapsulated PC12 and PC12A cells in terms of their ability to survive and produce catecholamines in vivo in a dopamine-depleted striatum, and (3) determine if BHK-GDNF, PC12, or PC12A cells reduce parkinsonian symptoms in a rodent model of Parkinson's disease. Capsules with BHK-GDNF or PC12 cells contained viable cells after 90 days in vivo, with little evidence of host tissue damage/gliosis. In rats with tyrosine hydroxylase (TH)-positive fibers remaining in the lesioned striatum, there was TH-positive fiber ingrowth into the membranes of the BHK-GDNF capsules. PC12-containing capsules had higher basal release of both dopamine and L-DOPA after 90 days in vivo than before implantation, while basal release of both dopamine and L-DOPA decreased in the PC12A-containing capsules. Both encapsulated PC12 and PC12A cells, but not encapsulated BHK-GDNF cells, decreased apomorphine-induced rotations. Parkinsonian symptoms (akinesia, freezing/bracing, sensorimotor neglect) related to the extent of dopamine depletion were evident even in rats with dopamine depletions of only 25%. Evidence that encapsulated cells may attenuate these parkinsonian symptoms was not detected but most of the rats were more severely depleted of dopamine than Parkinson's patients (less than 2% dopamine remaining in the entire striatum), and these tests were not sensitive to differences between rats with less than 10% dopamine remaining. These results suggest that cell encapsulation technology can safely provide site-specific delivery of dopaminergic agonists or growth factors within the CNS, without requiring suppression of the immune system, and without using fetal tissue. Of the three types of encapsulated cells examined in the present study, PC12 cells seem to offer the most therapeutic potential in rats with severe dopamine depletions.

Glial cell line-derived neurotrophic factor (GDNF), a new neurotrophic factor for motoneurones.

Glial cell line-derived neurotrophic factor (GDNF) has been postulated to be a specific dopaminergic neurotrophic factor since it selectively enhances the survival of dopaminergic neurones in vitro. We report here that GDNF can also act as a neurotrophic factor for motoneurones. GDNF released by GDNF-transfected BHK cells increases the activity of choline acetyltransferase (ChAT) in cultures from embryonic rat ventral mesencephalon containing cholinergic neurones from cranial motor nuclei and in cultured spinal motoneurones. Furthermore, local application of polymer-encapsulated BHK cells releasing GDNF to transected facial nerve in newborn rats diminishes the death of motoneurones normally occurring after axotomy in the neonatal period. The present results indicate that GDNF may have a therapeutic potential in human motoneurone diseases such as amyotrophic lateral sclerosis.

Implantation of polymer-encapsulated human nerve growth factor-secreting fibroblasts attenuates the behavioral and neuropathological consequences of quinolinic acid injections into rodent striatum.

Delivery of neurotrophic molecules to the central nervous system has gained considerable attention as a potential strategy for the treatment of neurological disorders. In the present study, a DHFR-based expression vector containing the human nerve growth factor gene (hNGF) was transfected into a baby hamster fibroblast cell line (BHK). Using an immunoisolatory polymeric device, encapsulated BHK-control cells and those secreting hNGF (BHK-hNGF) were transplanted unilaterally into rat lateral ventricles. Three days later, the same animals received unilateral injections of quinolinic acid (QA, 225 nmol) or the saline vehicle into the ipsilateral striatum. Approximately 2 weeks following surgery, animals were tested for apomorphine-induced rotation behavior. Animals which received BHK-hNGF cells rotated significantly less than those animals receiving BHK-control cells or QA alone. Histological analysis 29-30 days following capsule implantation demonstrated that BHK-hNGF cells attenuated the extent of host neural damage produced by QA as assessed by a sparing of ChAT- and NADPH-d-positive neurons. Moreover, a lessened GFAP reaction was apparent within the striatum of animals receiving BHK-hNGF cells. As measured by ELISA, hNGF was released by the encapsulated BHK-hNGF cells prior to implantation and following removal. Morphology of retrieved capsules revealed numerous viable and mitotically active BHK cells. These results suggest that implantation of polymer-encapsulated hNGF-releasing cells can be used to protect neurons from excitotoxin damage.

The aged monkey basal forebrain: rescue and sprouting of axotomized basal forebrain neurons after grafts of encapsulated cells secreting human nerve growth factor.

Six Rhesus monkeys between 24 and 29 years of age received unilateral transections of the fornix. Three monkeys then received intraventricular transplants of polymer-encapsulated baby hamster kidney (BHK) fibroblasts that had been genetically modified to secrete human nerve growth factor (hNGF). The remaining three monkeys received identical grafts except the cells were not modified to secrete hNGF. Monkeys receiving the fornix transection and control grafts displayed extensive reductions in the number of choline acetyltransferase- (57-75%) and p75 NGF receptor- (53%) immunoreactive medial septal neurons ipsilateral to the lesion/implant. In contrast, monkeys receiving transplants of encapsulated hNGF-secreting cells display only a modest loss of choline acetyltransferase- (0-36%) and p75 NGF receptor-(7-22.4%) immunoreactive septal neurons. Additionally, all monkeys receiving the hNGF-secreting implants, but none receiving control implants, displayed robust sprouting of cholinergic fibers within the septum ipsilateral to the transplant. Just prior to sacrifice, the capsules were retrieved and found to contain viable BHK cells releasing biologically relevant levels of hNGF. These data demonstrate that hNGF can provide trophic and tropic influences to aged primate basal forebrain neurons undergoing lesion-induced degeneration, supporting the contention that hNGF may prevent the degeneration of basal forebrain neurons in Alzheimer disease.

Visualization and ablation of phenylethanolamine N-methyltransferase producing cells in transgenic mice.

We cloned and sequenced the mouse phenylethanolamine N-methyltransferase (PNMT) gene which encodes the enzyme that catalyses the conversion of norepinephrine to epinephrine. The ability of various length sequences flanking the mouse or human PNMT genes to direct expression of reporter genes in transgenic mice was examined. We show that 9 kb of 5' flanking sequences from the cloned mouse PNMT gene can direct expression of the Escherichia coli beta-galactosidase (lacZ) gene to predicted regions of the adrenal, eye and brain in the adult transgenic mouse. The transgene was also expressed during development, in the myelencephalon, adrenal medulla and dorsal root ganglia. PNMT-producing cells were ablated by expression of the diphtheria toxin (DT-A) gene driven by the human PNMT promoter, resulting in abnormalities in the adrenal medulla, eye and testis. The hPNMT8 kb-DT-A line presents a model with which to examine the developmental ramifications of deletion of PNMT-producing cell populations from the adrenal medulla and retina.

Implants of polymer-encapsulated human NGF-secreting cells in the nonhuman primate: rescue and sprouting of degenerating cholinergic basal forebrain neurons.

Baby hamster kidney (BHK) cells were genetically modified to secrete high levels of human nerve growth factor (BHK-hNGF). Following polymer encapsulation, these cells were implanted into the lateral ventricle of four cynomolgus monkeys immediately following a unilateral transection/aspiration of the fornix. Three control monkeys received identical implants, with the exception that the BHK cells were not genetically modified to secrete hNGF and thus differed only by the hNGF construct. One monkey received a fornix transection only. All monkeys displayed complete transections of the fornix as revealed by a comprehensive loss of acetylcholinesterase-containing fibers within the hippocampus ipsilateral to the lesion. Control monkeys that were either unimplanted or received BHK-control (non-NGF secreting) cell implants did not differ from each other and displayed extensive losses of choline acetyltransferase and p75 NGF receptor (NGFr)-immunoreactive neurons within the medial septum (MS; 53 and 54%, respectively) and vertical limb of the diagonal band (VLDB; 21 and 30%, respectively) ipsilateral to the lesion. In contrast, monkeys receiving implants of BHK-hNGF cells exhibited a only a modest loss of cholinergic neurons within the septum (19 and 20%, respectively) and VLDB (7%). Furthermore, only implants of hNGF-secreting cells induced a dense sprouting of cholinergic fibers within the septum, which ramified against the ependymal lining of the ventricle adjacent to the transplant site. Examination of the capsules retreived from monkeys just prior to their death revealed an abundance of cells that produced detectable levels of hNGF in a sufficient concentration to differentiate PC12A cells in culture. These findings support the use of polymer-encapsulated cell therapy as a potential treatment for neurodegenerative diseases such as Alzheimer disease where basal forebrain degeneration is a consistent pathological feature. Moreover, this encapsulated xenogeneic system may provide therapeutically effective levels of a number of neurotrophic factors, alone or in combination, to select populations of neurons within the central nervous system.

Hypomyelinating peripheral neuropathies and schwannomas in transgenic mice expressing SV40 T-antigen.

We have prepared transgenic mice carrying a temperature-sensitive mutant of the SV40 oncogene (tsA-1609) under the control of 5' flanking sequences from the Schwann cell-specific P0 gene. Four of six founder mice showed moderate to severe hypomyelination in peripheral nerves of tail biopsies, with only rare myelinated fibers. Offspring were obtained from three of these founders. Northern blot and immunohistochemical analyses showed that expression of T-antigen was restricted to the PNS. Mice expressing the highest levels of T-antigen exhibited the most severe hypomyelination. Mice expressing lower levels developed transient mild hypomyelination, but after long latencies developed sporadic schwannomas. An immortalized cell line exhibiting properties of Schwann cells at an arrested stage of differentiation, termed "SCT-1," was derived from one of these tumors.

Polymer-encapsulated cells genetically modified to secrete human nerve growth factor promote the survival of axotomized septal cholinergic neurons.

Effective treatments for neurodegenerative disorders are limited by our inability to alter the progression of the diseases. A number of proteins have specific neuroprotective activities in vitro; however, the delivery of these factors into the central nervous system over the long term at therapeutic levels has been difficult to achieve. BHK cells engineered to express and release human nerve growth factor were encapsulated in an immunoisolation polymeric device and transplanted into both fimbria-fornix-lesioned rat brains and naive controls. In the lesioned rat brain, chronic delivery of human nerve growth factor by the encapsulated BHK cells provided nearly complete protection of axotomized medial septal cholinergic neurons. Human nerve growth factor continued to be released by encapsulated cells upon removal from the aspirative site after 3 weeks or from normal rat striatum after 3 and 6 months in vivo. Long-term encapsulated cell survival was confirmed by histologic analysis. This encapsulated xenogeneic system may provide therapeutically effective amounts of a number of neurotrophic factors, alone or in combination, to virtually any site within the body.

Development of myelin mosaicism in the optic nerve of heterozygotes of the X-linked myelin-deficient (md) rat mutant.

The oligodendrocyte population of the optic nerve has been suggested to arise either from a radial migration of neuroepithelial cells of the optic stalk or from the longitudinal migration of progenitor cells into the optic nerve from the brain, via the optic chiasm. Female heterozygotes of the X-linked myelin deficient (md) rat trait show a marked mosaic pattern of myelination of the optic nerves. This degree of mosaicism is not observed in other parts of the central nervous system. A total of 235 optic nerves from female rats of from 3 weeks to 24 months of age were examined by light microscopy. Nerves (104), from rats of all ages, showed defects in myelination consisting of distinct patches of non-myelination, often sharply demarcated from adjacent areas of normally myelinated axons. In some animals the abnormality was grossly apparent as areas of transparency within the intact optic nerves. In the majority of optic nerves showing mosaicism, defects in myelination were observed along the whole length of the nerve. However, a worsening of the defect toward the retinal end of the nerve was noted in 15 optic nerves, and an additional 11 nerves showed a defect in this region alone. It was also found that a number of rats had mosaicism in only one optic nerve. The preferential involvement of the retinal end of the optic nerves, and the asymmetrical involvement of the optic nerves within individual rats, is interpreted as indirect evidence in support of the proposed longitudinal migration of the oligodendrocyte precursor into the optic nerve.