Biomedical subjects

H L Vahlsing

Publications and source records attributed to H L Vahlsing.

At least 19 recordsLinked to original sources

PubMed1997

Spatial-temporal patterns of gene expression in mouse skeletal muscle after injection of lacZ plasmid DNA.

Gene therapy for muscular diseases requires the efficient transfection of a large proportion of myofiber cells within a given muscle. In the present experiments, patterns of beta-galactosidase expression were examined in mouse rectus femoris muscles at various time-points after a single injection of lacZ encoded plasmid DNA. beta-Galactosidase expression was detected 3 h after injection and rose to peak levels at 3-14 days, and then stabilized at lower levels. beta-Galactosidase staining was detected in an average of about 6% (up to 15%) of the total 4000 myofiber cells, and in about 70% of those myofibers located in the discrete area containing the greatest proportion of transfected cells. Soon after injection of DNA encoding cytoplasmic or nuclear-targeted beta-galactosidase, expression was noted predominantly in the myotendinous junction areas, after which beta-galactosidase activity progressed toward the central parts of the myofibers. This preferential transgene expression at the myotendinous junction may result from some unique, local property of the myofiber cells and/or from a restricted diffusion or binding of the injected plasmid DNA at tendinous surfaces. A better understanding of the reasons for this pattern of reporter gene expression in muscle may suggest procedures for increasing the number of myofiber cells transfected by direct DNA injections.

Animals↗

PubMed1996

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.

Animals↗

PubMed1995

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.

Animals↗

PubMed1994

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.

Animals↗

PubMed1991

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.

Animals↗

PubMed1991

Nerve growth factor promotes CNS cholinergic axonal regeneration into acellular peripheral nerve grafts.

Peripheral nerve grafts promote vigorous regeneration of adult mammalian CNS axons. Elimination of nerve-associated cells by freeze-thawing abolishes this promoting quality, possibly by creating inhibitory cellular debris and/or destroying the production of stimulatory factors by living Schwann or other cells. Here, debris-free acellular peripheral nerve segments placed between the disconnected septum and the hippocampal formation acquired almost no cholinergic axons after 1 month. However, such acellular nerve grafts treated before implantation with purified beta-nerve growth factor (NGF) contained nearly as many longitudinally oriented cholinergic axons as did fresh cellular nerve grafts. These results suggest that (i) NGF is required for the regeneration of adult CNS cholinergic axons into nerve grafts and (ii) an important function of living cells within peripheral nerve may be the production of neuronotrophic factors such as NGF.

Acetylcholinesterase↗

PubMed1990

Septohippocampal cholinergic axonal regeneration through peripheral nerve bridges: quantification and temporal development.

Axons of the adult mammalian CNS have been shown to regrow vigorously into peripheral nerve grafts. Using a cholinergic septohippocampal model for adult CNS regeneration, involving complete denervation of the hippocampal formation from its basal forebrain cholinergic afferents, this study has established quantitative parameters and a temporal baseline of cholinergic fiber regeneration into the dorsal hippocampal tissue through a peripheral sciatic nerve graft. In nerve-implanted animals (i) the nerve grafts are maximally invaded by AChE-positive fibers between 2 weeks and 1 month postlesion, (ii) the fibers entering the hippocampal formation from the graft show a peak numerical increase and rate of elongation around the first month and/or in the proximal hippocampal region, (iii) an apparently normal innervation pattern and fiber density in the most rostral 1.5 mm of the dorsal hippocampal formation is reached by 6 months postlesion. The present study provides a basis for future quantitative comparisons of manipulations of different components of the system, e.g., the contributing neurons, the bridging material, and the receiving central nervous tissue. The temporal/spatial pattern of fiber regeneration suggests that the hippocampal CNS tissue can be a good axonal growth-promoting environment, albeit with temporal and/or spatial limitations, and is therefore not an immutably restrictive environment for axonal regeneration.

Acetylcholinesterase↗

PubMed1990

Nerve growth factor infusion into the denervated adult rat hippocampal formation promotes its cholinergic reinnervation.

The well-documented but little-understood failure of lengthy axonal regeneration after injury of the adult mammalian CNS may be caused by an insufficient availability of local growth-promoting factors. If so, identifying and supplying the missing factors may result in better central axonal regeneration. This hypothesis was tested in an adult rat CNS model in which peripheral nerve grafts were placed into a lesion cavity between the septum and hippocampal formation. Continuous infusion of nerve growth factor (NGF) into the dorsal hippocampal tissue dramatically enhanced and accelerated the regrowth and penetration of cholinergic axons into the hippocampal formation. Thus, NGF can overcome the apparent resistance of the hippocampal CNS tissue to cholinergic reinnervation.

Acetylcholinesterase↗

PubMed1989

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.

Animals↗

PubMed1989

An improved device for continuous intraventricular infusions prevents the introduction of pump-derived toxins and increases the effectiveness of NGF treatments.

The recent demonstrations of the ability of nerve growth factor (NGF) to protect and promote the welfare of certain cholinergic neurons in the adult CNS have increased the need for safe, accurate, and reliable procedures for intracerebral administration of protein and other experimental agents. Osmotic minipumps have been used to infuse NGF into the lateral ventricle of adult rats, but a sustained and harmless performance of such infusions has not been fully evaluated. The study reported here has led to (i) the recognition that cytotoxic substances, released from some minipumps into the infusion fluid, may be responsible for various degrees of periventricular tissue damage, and (ii) the redesigning of an infusion device which, among other modifications, uses the osmotic pump to propel infusion fluid into the ventricle but prevents pump-derived materials from entering the infusate itself. Besides several other advantages, the modified infusion device has permitted the demonstration that NGF can fully protect experimentally axotomized medial septum cholinergic neurons and can do so with less variability than previously observed and without creating tissue damage.

Animals↗

PubMed1989

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.

Animals↗

PubMed1988

Fibroblast growth factor effects on peripheral nerve regeneration in a silicone chamber model.

We have developed a silicone nerve regeneration chamber that is partitioned into two compartments by a strip of nitrocellulose paper. The modified two-compartment chamber allows the investigation of the effects on rat sciatic nerve regeneration of trophic or growth factors that are initially bound to the nitrocellulose partition. In this study we compared the effects of untreated nitrocellulose, a siliconized nitrocellulose strip, and a strip that had been soaked in a basic fibroblast growth factor (FGF) solution. FGF is a known angiogenic factor and a mitogen for endothelial cells, fibroblasts, and Schwann cells. All of these cell types are present in the peripheral nerve. In vitro analyses, using 3T3 cells as test cells, showed that some of the bound FGF remained active on the nitrocellulose paper for at least 8-10 days. In vivo experiments, examined at 16 days post-implantation, revealed that spatial migration of all cellular elements (perineurial-like cells, vasculature, and Schwann cells) across the chamber gap was slower with untreated nitrocellulose strips than with siliconized strips but was most advanced with FGF-treated ones. Most striking was the well-developed vascular arborization of the regenerate within the FGF chambers. Histologic sections from the proximal one-half of the chamber revealed that the regenerate in untreated strip chambers consisted of fibrin matrix and erythrocytes, whereas a well-developed structure with all the cellular elements of a regenerating nerve was seen in several of the FGF strip chambers. We conclude that FGF stimulates peripheral nerve regeneration in this model.

Animals↗

PubMed1988

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.

Animals↗

PubMed1988

A two-compartment modification of the silicone chamber model for nerve regeneration.

In the nerve regeneration silicone chamber model, the regenerate which forms across a 10-mm gap between proximal and distal nerve stumps is a monofascicular structure with an outer perineurial-like cell sheath. Recent work has provided indications that the geometry of the regenerate within a silicone chamber can be altered by experimental modifications of the chamber matrix. In the present study we modified the standard silicone chamber into a two-compartment chamber by inserting a 6- or 10-mm-long siliconized nitrocellulose strip in order to obtain two separate regenerates. Light microscopy 16 days after implantation revealed that two separate nerve structures had formed, one on each side of the nitrocellulose partition and adjacent to it, and each with its own perineurial-like cell sheath. In chambers with 6-mm-long strips a monofascicular regenerate started from the proximal stump and divided into two separate structures as it approached the proximal end of the strip: the two fascicles joined again into a monofascicular structure in the distal portion of the chambers. The new two-compartment silicone chamber model appears suitable for future examinations of experimental fasciculation. In addition, the nitrocellulose partition should allow one to study specific effects of growth factors on axonal regeneration in vivo, as growth factors bind strongly to untreated nitrocellulose while retaining their biological activity.

Animals↗

PubMed1987

A small-gauge cannula device for continuous infusion of exogenous agents into the brain.

A method is described for the construction of an intraventricular or intraparenchymal cannula device, which when connected to an Alzet osmotic pump, can be used for the continuous infusion of experimental solutions into the brain. A 33-gauge, stainless-steel cannula is encased within a dental acrylic stabilization platform prior to stereotaxic implantation, and after implantation, the platform is glued to the animal's skull using cyanoacrylate adhesive. This procedure provides for the long-term stability (at least 4 weeks) of the small-gauge cannula without the need for additional stabilization skull screws, thus minimizing damage to surrounding tissues by the cannula and postsurgical trauma to the animal. Using the stock model 2002 Alzet pump to infuse artificial cerebral spinal fluid at a flow rate equal to 0.5 microliter/h, an inflammatory tissue reaction around the cannula tip was consistently found after 2 weeks of continuous intraparenchymal infusion. However, the inflammatory reaction could be significantly reduced or eliminated by decreasing the flow rate to approximately 0.25 microliter/h, using a modified Alzet pump. Alternatively, the stock 0.5 microliter/h pump could be used without causing parenchymal damage if the cannula tip was implanted into the lateral ventricle.

Animals↗

PubMed1987

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.

Animals↗

PubMed1986

Long-term effects of spinal cord transection on fast and slow rat skeletal muscle. I. Contractile properties.

Contractile properties of rat soleus and extensor digitorum longus muscles were studied 1 year after complete thoracic spinal cord transection (spinal cord level T9). Force-generating capacity and contraction speed were unchanged in the extensor digitorum longus 1 year after transection. However, the rate of contraction and relaxation increased in the soleus as reflected by a decrease in time-to-peak tension and increase in fusion frequency. Additionally, the soleus muscle cross-sectional area decreased significantly (50%) while generating the same absolute tension. Thus, a large increase in soleus specific tension (force per unit area) was observed. These data, in conjunction with the increase in contractile speeds, suggest soleus slow-to-fast fiber type conversion secondary to cordotomy. Discriminant analysis of the contractile properties yields fusion frequency as the best discriminator between muscle groups. Thus, following cordotomy, predominantly slow muscles are affected to a greater extent than fast muscles.

Analysis of Variance↗

PubMed1986

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.

Amputation, Surgical↗