Dental abnormalities in rats after a single large dose of cyclophosphamide.

Delayed drug-related mortality in rats treated with a single high dose (75 mg/kg) of cyclophosphamide complicated experiments using this drug treatment. We observed that this delayed mortality was due to dental abnormalities including broken teeth, absent teeth, extra long teeth, and/or supernumerary teeth. These dental abnormalities developed about 140 days after treatment and, if left untreated, interfered with eating. Eventually, the untreated rats starved. Clipping their long teeth and feeding the rats powdered chow eliminated the deaths. Researchers should be aware that high doses of cyclophosphamide may result in dental abnormalities several months after the treatment.

Cancer gene therapy using plasmid DNA: safety evaluation in rodents and non-human primates.

To evaluate the safety of a plasmid DNA-lipid complex, a series of good laboratory practice (GLP) safety studies were conducted with VCL-1005, a plasmid DNA expression vector containing both the human class I MHC HLA-B7 heavy-chain and the beta 2-microglobulin (beta 2m) light-chain genes formulated with the cationic lipid, DMRIE/DOPE. In mice, the repeated intravenous injection of VCL-1005 at plasmid DNA doses of 0.1, 1.0, or 10 micrograms for 14 days had only incidental effects on clinical chemistry and hematology, and did not result in any organ pathology. Repeated intrahepatic injections of VCL-1005 in mice did not result in significant liver histopathology or significant alterations in liver enzymes. In cynomolgus monkeys, the repeated intravenous administration of VCL-1005 at a cumulative dose of 720 micrograms of DNA had no effects on clinical chemistry, hematology, or organ pathology. Thus, systemic administration of a plasmid DNA expression vector containing the coding sequence for a foreign MHC class I molecule did not result in significant toxicity or a pathological immune response in animals. These results suggest that the direct transfer of VCL-1005, a plasmid DNA-lipid complex, could be used for the safe in vivo delivery of recombinant DNA for a cancer gene therapy trial.

An improved plasmid DNA expression vector for direct injection into skeletal muscle.

In previous work, the direct injection of 50 micrograms of a plasmid DNA vector encoding firefly luciferase (VR1205) into murine quadriceps muscle produced an average of 6.5 ng of luciferase per muscle at 7 days postinjection. In this report, various elements of the VR1205 vector were modified to increase gene expression levels or to eliminate undesired viral sequences. Expression of the modified vectors was then compared to VR1205 using the intramuscular injection assay. In general, modifications to promoter, enhancer, and intronic sequences either decreased luciferase expression levels or had no effect. However, modifications to the polyadenylation and transcriptional termination sequences, plasmid backbone elements, and the luciferase gene itself each increased luciferase expression levels. The best-expressing vector, designated VR1255, contained a combination of these incrementally beneficial changes. A single intramuscular injection of 50 micrograms of VR1255 produced 300 ng of luciferase at 7 days postinjection, an expression level 46-fold higher than the VR1205 vector (or 22-fold higher, excluding modifications to the luciferase gene) and 154-fold higher than a commercially available luciferase expression vector. Thus, VR1255 represents an improved plasmid DNA vector that may be useful for gene therapy applications.

Cell death in Clarke's column after spinal cord transection.

The death of embryonic central nervous system (CNS) neurons deprived of a target is well established. In adult rats, similar cell death of corticospinal and rubrospinal motor neurons occurs as a delayed response to spinal cord transection. We document the loss of neurons in Clarke's column, secondary ascending spinocerebellar neurons in adult rats, after complete spinal cord transection at T-9. Twenty-five weeks after spinal cord transection, horseradish peroxidase (HRP) studies showed a dramatic loss of labeled cells in rats with transected spinal cords as compared to matched control rats. Cresyl echt violet-stained sections failed to support the hypothesis that unlabeled cells persist in a shrunken, inactive state; instead we found far fewer identifiable neurons in Clarke's column. Although we saw little gliosis in the area of cell loss, gliosis was evident in the adjacent corticospinal tract which was severed in the original surgical injury. Amputation of the right hind limb resulted in a paradoxical increase in labeled Clarke's column cells on the right. Total cells stained with cresyl echt violet in amputated animals were not different from right to left. The increase in labeled cells on the amputated side may have been caused by an increase in metabolic activity of these deafferentated neurons which resulted in more effective axoplasmic transport of the HRP label.

Changes in number and size of Clarke's column neurons after cord transection.

The number of large neurons in Clarke's column of the L-1 segment of the spinal cord of the rat decreases five or more weeks after a T-9 spinal cord transection. Analysis of cells at 1, 2, 3, 5, 7, 9, 12, and 15 weeks (wk) postoperatively demonstrates a loss of large neurons at each time interval beyond five wk postoperatively. Comparison of cell sizes found in the anatomic region of Clarke's column at two or three wk postoperatively with the cells found at 15 wk after transection and their respective control groups, shows a decrease in total cells found in operated rats 15 wk postoperative with a profound decrease in larger neurons in these rats. We did not detect a significant offsetting increase in smaller neurons. We believe the observed changes are due to death of large neurons and can find no evidence to support the contention that axotomized cells persist in a shrunken, atrophic state.

Fink-Heimer/Nauta demonstration of regenerating axons in the rat spinal cord.

Six months after complete spinal cord transection, regenerated descending motor axons can be found by electrophysiologic testing and can also be demonstrated anatomically using the Fink-Heimer/Nauta technique. Regeneration was found in all animals, treated or control, but statistically significant increased regeneration as measured by the Fink-Heimer/Nauta technique was found in animals treated with a single injection of 75 mg/kg of cyclophosphamide 24 hours after the original spinal cord transection. This result is similar to those of another study using the orthograde axonal transport of tritiated proline as a measure of axonal regeneration. The mode of action of cyclophosphamide may be by its immunosuppressive properties because this drug is most effective when administered 24 hours after cord transection, a time when its effects as an immunosuppressant would be maximal.

Retrograde transport in corticospinal neurons after spinal cord transection.

Complete spinal cord transection at T-6/T-7 in rats caused a decrease in the number of surviving corticospinal neurons. Cell death began 5 and 10 weeks after cord injury. The number of surviving cells decreased progressively for at least 25 weeks after injury. Surviving cells were identified by their ability to transport horseradish peroxidase (HRP) retrograde from a T-1/T-2 insertion site to cortical cell somas. Therapy aimed at promoting corticospinal tract regeneration must be started early after spinal cord injury.

Histologic evidence for death of cortical neurons after spinal cord transection.

Dying cortical neurons, identified by standard histologic criteria, were observed in the rat sensory/motor cortex after spinal cord transection. The peak incidence of these changes was 10 weeks after injury. At that time, spinal-cord-injured animals showed 10 times as many abnormal cells as were found in matched controls (p less than or equal to 0.05). Dying cells were found in the same anatomic location as corticospinal neurons axotomized by the experimental injury, and at the expected time after injury. The survival of corticospinal neurons in these young adult rats may be dependent on obtaining a crucial input from an appropriate target cell (neuron).

Recovery in rats after spinal cord injury.

Previous studies from this laboratory have shown evidence of regeneration of long descending spinal motor tracts in rats after spinal cord transection and treatment to modify the animals' immune response. In this study, less extensive surgical lesions were combined with the most favorable drug treatment (75 mg per kilogram of cyclophosphamide in a single dose) in an effort to improve the prospects for regeneration. Less than complete spinal cord transections in the rat were frequently followed by clinical and electrophysiologic evidence of return of function. Such return of function appears to depend on a reorganization of the nervous system that results in the use of the few remaining fibers to transmit motor information rather than on regeneration. Immunosuppressive treatment had no effect on these results.

Immunization with plasmid DNA using a pneumatic gun.

We characterize a method by which the Med-E-Jet pneumatic vaccination gun can be used to propel intact, supercoiled plasmid DNA through skin and into skeletal muscles of mice. Intramuscular injection of plasmids containing the firefly luciferase gene linked to the human cytomegalovirus promoter resulted in the expression of several hundred picograms of luciferase enzyme in quadriceps muscles. Intramuscular injections of a plasmid containing the influenza A nuclear protein gene regulated by the same promoter resulted in the generation of potent and specific anti-nuclear protein humoral and cellular immune responses. This convenient and rapid injection method would be well-suited for genetic immunization of humans.

Nerve growth factor effects on cholinergic neurons of neostriatum and nucleus accumbens in the adult rat.

Following intraventricular nerve growth factor infusion in adult rats, the choline acetyltransferase immunostaining of the neuropil and neuronal cell bodies of the neostriatum (caudate-putamen) and nucleus accumbens was more intense on the side of the infusion. Furthermore, the average cross-sectional size (micron2) of the cholinergic somata was increased by about 40 and 20% in the striatum and accumbens, respectively. This unilateral response could be elicited in intact rats as well as in rats receiving a prior aspirative transection of the fimbria-fornix. The reported lack of (low-affinity) nerve growth factor receptor immunostaining in these neurons suggests that the nerve growth factor effects are most likely transduced by high-affinity receptors. The ability of these apparently undamaged cholinergic interneurons to respond to exogenous nerve growth factor with an increase in choline acetyltransferase content and cell body size suggests that they are benefiting from a less-than-maximal support by endogenous nerve growth factor in the normal young adult rat.

Light microscopic, immunohistochemical localization of the pia-glial basal lamina.

The current histologic methods for studying the pia-glial basal lamina (BL) are inappropriate for high contrast, permanent light microscopy preparation. We have developed a staining technique for epithelial BL which is highly specific, extremely sensitive, permanent, relatively inexpensive, and suitable for light or electron microscopy (EM). Central nervous system (CNS) basement membrane zone (BMZ) antigens were isolated by the technique of Meezan (1975) from female albino Wistar rats. Using this CNS BMZ preparation as an antigenic source, a hyperimmune rabbit serum was developed. This serum was exhaustively adsorbed with rat splenic pulp to remove undesirable antibodies to endothelial BL and collagen. The peroxidase-antiperoxidase indirect antibody technique was used to test the staining specificity of this splenic adsorbed serum on different tissues containing BLs of known origin and/or function. The results indicated that this BL staining technique was specific for epithelial BL of the rat and of some other species.

Nerve growth factor (NGF) reverses axotomy-induced decreases in choline acetyltransferase, NGF receptor and size of medial septum cholinergic neurons.

Intraventricular nerve growth factor (NGF) infusion in the adult rat can prevent and also, if delayed, reverse the disappearance of most of the axotomized medial septum cholinergic neurons immunostained for choline acetyltransferase (ChAT). We have utilized the delayed NGF treatment protocol to (i) extend to 3 months the delay time between axotomy and NGF treatment, (ii) define the time course of their recovery, (iii) determine that immunostaining for the (lower affinity) NGF receptor (NGFR) parallels loss and reversal of the ChAT marker, and (iv) evaluate changes in cholinergic somal size following axotomy and subsequent NGF treatment. While NGF treatments starting only 7 days after the fimbria-fornix transection (axotomy) almost entirely restored the number of both ChAT- and NGFR-positive medial septum neurons, longer delayed (2-3 weeks) treatment brought about recovery from the baseline of 20-25% to only about 70% of the control numbers. This limited recoverability, however, persisted even after a 95 day delay period. In all cases examined maximal recoveries were achieved within 3-7 days of NGF treatment. Neuronal size analyses provided evidence for an axotomy-induced atrophy. NGF treatments, started with 1 or 2 week delays, not only reversed fully the average somal size loss but also induced an actual hypertrophy of several of those neurons. These results provide additional evidence that at least half of the apparent loss of cholinergic medial septum neurons upon axotomy is due to a loss of markers such as the transmitter-related enzyme ChAT and NGFR rather than to actual neuronal cell death. These results also show that NGF exerts a genuine trophic influence by regulating the size of its target neurons as well as their content of several proteins.

Dose-dependent responses to nerve growth factor by adult rat cholinergic medial septum and neostriatum neurons.

This study describes the relationship between the concentration of intraventricularly infused nerve growth factor (NGF) and several responses by axotomized cholinergic medial septum neurons and normal cholinergic neostriatal neurons of the adult rat. NGF infused for 14 days starting either immediately after a unilateral fimbria-fornix transection or after a 2-week delay period elicited similar dose-response relationships for the maintenance or restoration of ChAT and NGF receptor positivity and cell body size and for intraseptal 'sprouting' of the axotomized medial septum neurons. Thus, in the medial septum it appears that the expression of 'marker' molecules, cell body size and the induction of 'sprouting' are regulated by virtually the same concentrations of NGF in the two treatment strategies. This suggests that NGF has a general regulatory role and injured but untreated neurons remain fully susceptible to NGF at least up to 2 weeks after the lesion. A 14-day infusion with NGF also induced an above-normal cell body size (hypertrophy) both in axotomized medial septum and in intact striatal cholinergic neurons. The hypertrophic response of normal striatal neurons required less NGF than did that of medial septum neurons. Since the striatal response began to be detectable at a similar concentration as that required for the full maintenance or restoration of ChAT and NGF receptor positivity it could be seen as an unwanted side-effect. The definition of a sub-optimal dose with which a significant, but not maximal response can be elicited will allow future evaluations of potentially additive or synergistic actions by other agents.

Labeled corticospinal neurons one year after spinal cord transection.

One year after a T9 spinal cord transection, horseradish peroxidase was inserted into the spinal cord at T3-T4. Only about 7% of the number of corticospinal neurons labeled in control rats were labeled in exactly matched transected rats. This long-term loss of labeled neurons makes cell death the most likely explanation for the failure to identify corticospinal neurons in spinal-cord-transected rats.

Basal lamina at the site of spinal cord transection in the rat: an ultrastructural study.

This electron microscopic study confirms that basal lamina (BL) begins to cap the cut end of the spinal cord 15 days after spinal cord transection. BL is first seen immediately adjacent to reactive glial cells but only when there is collagen in the nearby interstitial space. This finding suggests that collagen may provide a trigger to initiate the production of BL by reactive glia. We found no direct evidence that BL in this injury area impeded the outgrowth of regenerating neurites.

Basal lamina at the site of spinal cord injury in normal, immunotolerant and immunosuppressed rats.

The cut ends of a rat spinal cord are capped with basal lamina (BL) within 20 days. This BL may block regenerating axons. BL at the transection site in rats made immunologically unresponsive to central nervous system antigens is not significantly different from that of control rats, but rats treated with cyclophosphamide show a less complete BL cap during the first 25 days. This may account for the increased axonal regeneration found in cyclophosphamide-treated rats.

Cyclophosphamide-induced abnormalities in the incisors of the rat.

A single injection of 75 mg/kg cyclophosphamide caused gross dental abnormalities in rats. Broken, malformed, overgrown, and "extra" incisors developed several weeks after drug treatment. Radioautographic investigations show no unusual features in the morphology or labeling with H3-thymidine in the odontogenic cells. The results suggest that the cytotoxic effect of the drug is a temporary one which induces a residual alteration of tooth growth.

Corticospinal neurons 25 weeks after right hind limb amputation.

Neuronal cell death in embryos and adult animals is seen after removal of target tissue. Transsynaptic cell death has been described in the mammalian visual system and suggested as a possible mechanism for loss of upper motor neurons in amyotrophic lateral sclerosis. We previously demonstrated that amputation of a hind limb decreased the number of motor neurons in the rat spinal cord. Careful counts of corticospinal neurons in these rats 25 weeks after amputation failed to demonstrate any loss of corticospinal neurons. Although amputation caused a loss of ventral horn neurons, no subsequent loss of upper motor neurons was detected at 25 weeks.

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 Alzheimer's dementia, which are characterized by chronic and progressive losses in the function of specific sets of neurons.