Mapping of microsatellite loci and association of aorta atherosclerosis with LG VI markers in the rabbit.

Twenty-three rabbit microsatellites were extracted from the EMBL nucleotide database. Nine of these markers, together with nine earlier published microsatellite markers, were found to be polymorphic between the AX/JU and IIIVO/JU inbred strains. By using an F(2) intercross we could integrate five markers into the rabbit linkage map. One anonymous microsatellite marker could be assigned to chromosome 1, and one microsatellite marker, located within the metallothionein-1 gene, could be added to linkage group VI (LG VI). Three microsatellite markers (one anonymous, one located within the PMP2 gene, and one located within the FABP6 gene) constitute a new linkage group (LG XI). We also measured the degree of dietary cholesterol-induced aorta atherosclerosis in the F(2) animals. A significant cosegregation was found between the degree of aorta atherosclerosis and the allelic variation of the biochemical marker Est-2 on LG VI in male rabbits. This association was not found in female rabbits.

Automated quantitative gait analysis during overground locomotion in the rat: its application to spinal cord contusion and transection injuries.

Analysis of locomotion is an important tool in the study of peripheral and central nervous system damage. Most locomotor scoring systems in rodents are based either upon open field locomotion assessment, for example, the BBB score or upon foot print analysis. The former yields a semiquantitative description of locomotion as a whole, whereas the latter generates quantitative data on several selected gait parameters. In this paper, we describe the use of a newly developed gait analysis method that allows easy quantitation of a large number of locomotion parameters during walkway crossing. We were able to extract data on interlimb coordination, swing duration, paw print areas (total over stance, and at 20-msec time resolution), stride length, and base of support: Similar data can not be gathered by any single previously described method. We compare changes in gait parameters induced by two different models of spinal cord injury in rats, transection of the dorsal half of the spinal cord and spinal cord contusion injury induced by the NYU or MASCIS device. Although we applied this method to rats with spinal cord injury, the usefulness of this method is not limited to rats or to the investigation of spinal cord injuries alone.

New assessment techniques for evaluation of posttraumatic spinal cord function in the rat.

To evaluate new pharmacologic agents with potentially beneficial effects on posttraumatic spinal cord function, we used a modified weight drop (WD) technique to induce spinal cord injuries. These contusive spinal cord injuries in the rat closely mimic the human clinicopathologic situation. Especially for drug screening purposes, the moderate and mild injuries are of interest, as both the beneficial and potentially harmful effects of experimental treatment can be detected. In this study, we describe two new functional tests that were particularly designed to detect small differences in spinal cord function after moderate and mild injuries. First, for examination of locomotion, a computer analysis of the thoracolumbar height (TLH) was designed. Second, for investigation of the conduction properties of the injured rat spinal cord, we measured rubrospinal motor evoked potentials (MEP). The efficacy of the new assessment techniques to monitor spinal cord function was compared to Tarlov scores and to morphometric analysis of preserved white matter at the injury site. The results of this study indicated that for behavioral analysis, TLH measurements as compared with Tarlov rating appeared to be more sensitive for exact and objective discrimination between small differences in motor function. Amplitudes of the rubrospinal MEP, but not latencies or the number of peaks, proved to be most sensitive to determine subtle differences in posttraumatic spinal cord function. A significant linear correlation was found between TLH and amplitude of the rubrospinal MEP. We conclude that for objective assessment of the spinal cord after moderate and mild contusive injury, TLH and rubrospinal MEP amplitudes are very valuable measures to demonstrate small functional differences.

Functional recovery after central infusion of alpha-melanocyte-stimulating hormone in rats with spinal cord contusion injury.

Melanocortins, peptides related to alpha-melanocortin-stimulating hormone (alpha MSH) and adrenocorticotropic hormone (ACTH), are known to improve axonal regeneration following peripheral nerve injury and stimulate neurite outgrowth from central nervous system (CNS) neurons both in vitro and in vivo. The neurite outgrowth promoting capacity of alpha MSH has prompted us to investigate the effects of intrathecal application of alpha MSH on functional and electrophysiological recovery in a well-characterized model of spinal cord contusion injury. Different doses of alpha MSH were applied via osmotic minipumps into the cisterna magna for 10 days, thereby delivering the peptide directly into the CNS. Functional recovery was monitored during 8 postoperative weeks by means of the Basso, Beattie, and Bresnahan locomotor rating scale, and the thoracolumbar height test. At the end of the study, electrophysiological analysis of rubrospinal motor evoked potentials as performed. Our data showed that application of 3.75 micrograms/kg/h alpha MSH resulted in a marked functional recovery, accompanied by a decrease in the latency of the rMEP. This study demonstrates that intrathecal application of alpha MSH results in functional recovery after spinal cord contusion injury. These findings may initiate new treatment strategies and/or the use of melanocortins in human spinal cord injury.

Effects of enriched housing on functional recovery after spinal cord contusive injury in the adult rat.

To date, most research performed in the area of spinal cord injury focuses on treatments designed to either prevent spreading lesion (secondary injury) or to enhance outgrowth of long descending and ascending fiber tracts around or through the lesion. In the last decade, however, several authors have shown that it is possible to enhance locomotor function after spinal cord injury in both animals and patients using specific training paradigms. As a first step towards combining such training paradigms with pharmacotherapy, we evaluated recovery of function in adult rats sustaining a spinal cord contusion injury (MASCIS device, 12.5 mm at T8), either housed in an enriched environment or in standard cages (n = 15 in both groups). The animals in the enriched environment were stimulated to increase their locomotor activity by placing water and food on opposite sides of the cage. As extra stimuli, a running wheel and several other objects were added to the cage. We show that exposure to the enriched environment improves gross and fine locomotor recovery as measured by the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale, the BBB subscale, the Gridwalk, and the Thoracolumbar height test. However, no group differences were found on our electrophysiological parameters nor on the amount of spared white matter. These data justify further studies on enriched housing and more controlled exercise training, with their use as potential additive to pharmacological intervention.

Combined treatment with alphaMSH and methylprednisolone fails to improve functional recovery after spinal injury in the rat.

To date, relatively little progress has been made in the treatment of spinal cord injury (SCI)-related neurological impairments. Until now, methylprednisolone (MP) is the only agent with clinically proven beneficial effect on functional outcome after SCI. Although the mechanism of action is not completely clear, experimental data point to protection against membrane peroxidation and edema reduction. The melanocortin melanotropin is known to improve axonal regeneration following sciatic nerve injury, and to stimulate corticospinal outgrowth after partial spinal cord transection. Recently, we showed that intrathecally administered alphaMSH had beneficial effects on functional recovery after experimental SCI. Since both drugs have shown their value in intervention studies after (experimental) spinal cord injury (ESCI), we decided to study the effects of combined treatment. Our results again showed that alphaMSH enhances functional recovery after ESCI in the rat and that MP, although not affecting functional recovery adversely by itself, abolished the effects observed with alphaMSH when combined. Our data, thus, suggest that the mechanism of action of MP interferes with that of alphaMSH.

Local application of collagen containing brain-derived neurotrophic factor decreases the loss of function after spinal cord injury in the adult rat.

We studied the effect of local application of brain-derived neurotrophic factor (BDNF) on functional recovery after dorsal spinal cord transection in the adult rat. BDNF was applied at the site of the lesion in rat tail collagen type I. Locomotion was measured for 4 weeks using the BBB locomotor rating scale. One day after injury and application of BDNF the performance of treated rats was significantly increased as compared to controls (BBB-score 11.5+/-1.3 (mean +/- SEM) and 7.5+/-1.3, respectively). This difference remained significant during the first week. Histological examination of the spared spinal cord tissue at the lesion centre 4 weeks after lesioning showed no significant difference between control and BDNF-treated animals. The results indicate that local application of BDNF results in a decreased loss of function in the partially transected rat spinal cord starting one day after injury.

Beneficial effects of the melanocortin alpha-melanocyte-stimulating hormone on clinical and neurophysiological recovery after experimental spinal cord injury.

OBJECTIVE: Melanocortins, peptides related to melanocyte-stimulating hormone (MSH) and corticotropin (ACTH), exhibit neurotrophic and neuroprotective activity in several established models of peripheral and central nervous system damage. The beneficial effects of melanocortins on functional recovery after experimental brain damage and central demyelinating diseases have prompted us to investigate alpha MSH treatment in a weight drop model of traumatic spinal cord injury in rats. METHODS: In two independent randomized blinded experiments, treatment with either alpha MSH (75 micrograms/kg of body weight administered subcutaneously every 48 h for 3 weeks after trauma) or single high-dose (30 mg/kg, 30 min after injury) methylprednisolone was compared with saline treatment in rats subjected to a moderately severe 20-gcm weight drop injury. Spinal cord function was monitored using behavioral, electrophysiological, and histological parameters. RESULTS: In both experiments, alpha MSH significantly improved recovery, as illustrated by Tarlov scores, thoracolumbar height, and amplitude of rubrospinal motor evoked potentials. The magnitude of the alpha MSH effect on motor performance was comparable with the one observed after treatment with methylprednisolone. CONCLUSION: The reproducible neurological and electrophysiological improvement in spinal cord function of animals treated with alpha MSH suggests a new lead in the treatment of traumatic spinal cord injury.

Role of DL-lactic acid as an intermediate in rumen metabolism of dairy cows.

The role of DL-lactic acid as an intermediate in the rumen of a Friesian X Holstein dairy cow adapted to a diet of hay ad libitum plus 12 kg of a concentrate mixture was studied in vitro and in vivo. Concentrations of soluble sugars in the rumen fluid became maximal at 30 min postfeeding, but at 90 min no sugars were detectable. The DL-lactate concentration increased very rapidly to about 30 mm at 30 min after feeding, whereas the maximum total VFA concentration was reached 15 min later. More than 80% of the DL-lactate fermented to VFA was converted by Megasphaera elsdenii. Whereas only 16% of L-lactate was fermented to propionate, 75% of the D-lactate was converted to propionic acid. When all soluble sugars had been fermented, the participation of M. elsdenii to lactate fermentation declined and fermentation patterns for D- and L-lactate became similar yielding mostly acetate. Except for a brief period immediately after feeding, DL-lactate did not appear to be an important precursor of VFA in the rumen of a cow adapted to concentrate feeding. DL-lactate may become a more important intermediate in rumen fermentation temporarily when dairy cows are gradually changed from a hay diet to a diet including concentrates. The first 30 d after parturition, when the changeover takes place, is an unstable period, during which the microbial population is changing to fit the new environment.

Segregation of genes from donor strain during the production of recombinant congenic strains.

Recombinant congenic strains (RCS) constitute a set of inbred strains which are designed to dissect the genetic control of multigenic traits, such as tumour susceptibility or disease resistance. Each RCS contains a small fraction of the genome of a common donor strain, while the majority of genes stem from a common background strain. We tested at two stages of the inbreeding process in 20 RCS, derived from BALB/cHeA and STS/A, to see whether alleles from the STS/A donor strain are distributed over the RCS in a ratio as would theoretically be expected. Four marker genes (Pep-3; Pgm-1; Gpi-1 and Es-3) located at 4 different chromosomes were selected and the allelic distribution was tested after 3-4 and after 12 generations of inbreeding. The data obtained do not significantly deviate from the expected pattern, thus supporting the validity of the concept of RCS.

'Normalization' of germfree mice after direct and indirect contact with mice having a 'normal' intestinal microflora.

Germfree mice were associated via direct and indirect contact with a 'normal' microflora by placing 'normal' mice in an isolator with germfree mice. Relative caecal weights, the ratio of secondary to primary bile acids, the presence of filamentous segmented bacteria in the small intestine and faecal beta-aspartylglycine were normal 5 days after direct contact and 15 days after indirect contact. Enterobacteriaceae were demonstrated by the third day after direct contact and the fourth day after indirect contact. Volatile and non-volatile fatty acids in the caecal contents were variable and appeared to be unrelated to the 'normalization' process of germfree mice after association with a microflora.

Strain-specific response to hypercholesterolaemic diets in the rat.

The cholesterolaemic effect of 2 hypercholesterolaemic diets was tested in 12 rat inbred strains. Diet I is a commercial diet supplemented with 2.0% (w/w) cholesterol and 5.0% (w/w) olive oil; diet II is identical to diet I with addition of 0.5% (w/w) sodium cholate. Strains with the highest plasma cholesterol response after diet I (BN and LEW) also had the highest cholesterol response after diet II (hyperresponders, mean response > 3.5 mmol/l). In the strains DA, SHR, BC, WAG, LOU, PVG and BUF the strain mean cholesterol response remained below 1.3 mmol/l after both diets (hyporesponders). Strains F344 and OM had an intermediate cholesterol response after both diets (normoresponders, mean response between 1.3 and 3.5 mmol/l). Only in the strains LOU, PVG and SHR there appeared to be a significant higher cholesterol response after diet II when compared with the cholesterol response after diet I. In the strain WKY this difference was of a borderline significance (P = 0.052) and this strain turned from a normoresponder after diet I into a hyperresponder after diet II. Liver cholesterol levels as measured after feeding diet II for two weeks also appeared to be strain-specific. No correlation was found between the plasma cholesterol response after diet II and the liver cholesterol levels. Changes in plasma phospholipid and triglyceride levels have been measured for both diet I and diet II. For group means a correlation between the cholesterol response and the change in phospholipid levels was found (r = 0.86 for diet I, P < 0.001 and r = 0.76 for diet II, P < 0.01). No such correlation was found for triglyceride levels.

The 'normalization' of germ-free guineapigs with host-specific caecal microflora.

Hysterectomy-derived germ-free guineapigs were given colonization-resistant caecal flora from mice (mCRF) or microflora obtained from the caecum of an antibiotic-decontaminated conventional guineapig (gpCRF) and compared with guineapigs raised conventionally with the sow. Body weight and the following intestinal parameters were determined for the groups: colonization resistance (CR) to Escherichia coli, relative caecal weight (RCW), beta-aspartylglycine (faeces), volatile fatty acids (caecum) and bile acids (faeces). mCRF guineapigs showed values quite different from control animals for CR and RCW, indicating the unsuitability of mouse CRF for normalizing guineapigs. In gpCRF guineapigs CR and RCW values were comparable with controls, indicating the suitability of the guineapig flora for normalizing guineapigs. mCRF guineapigs housed with gpCRF guineapigs, showed an improvement in CR and RCW, yielding values found in control animals.

The 'normalization' of germ-free rabbits with host-specific caecal microflora.

Hysterectomy-derived germ-free rabbits were given colonization-resistant caecal flora (CRF) from mice, or microflora obtained from the caecum of an antibiotic-decontaminated conventional rabbit and compared with rabbits conventionally raised with the doe. Bodyweight and the following intestinal parameters were determined for the 3 groups: colonization resistance to E. coli, relative caecal weight, villus:crypt ratio (ileum), beta-aspartylglycine (faeces), volatile fatty acids (caecum), and bile acids (faeces). Germ-free rabbits given mouse CRF-flora showed values quite different from control animals for most parameters, indicating unsuitability of mouse CRF flora to 'normalize' rabbits. In germ-free rabbits given modified (antibiotic-treated) rabbit flora, values for most parameters were intermediate between those found for the other 2 groups. This species-specific caecal flora should be improved to provide full 'normalization' of germ-free rabbits.

Intraspinal administration of an antibody against CD81 enhances functional recovery and tissue sparing after experimental spinal cord injury.

We previously demonstrated that the tetraspanin protein CD81 is up-regulated by astrocytes and microglia after traumatic spinal cord injury in rats and that CD81 is involved in adhesion and proliferation of cultured astrocytes and microglia. Since these reactive glial cells contribute to secondary damage and glial scar formation, we studied the effect of local administration of an anti-CD81 antibody in experimental spinal cord injury. Adult rats were subjected to a moderate spinal cord contusion injury and treated for 2 weeks with different doses of the anti-CD81 antibody AMP1 (0.5-5 microg/h) or non-immune IgG (5.0 microg/h). A technique was developed to infuse the antibodies directly into the lesion site via an intraspinal cannula connected to a pump. Functional recovery was monitored during 8 postoperative weeks by means of the Basso, Beattie and Bresnahan (BBB) locomotor rating scale, the BBB subscore and Grid-walk test. At the end of the study, quantitative histology was performed to assess tissue sparing. Our data showed that by itself cannulation of the lesion site resulted in minimal functional and histological impairments. Application of 0.5 microg/h AMP1 resulted in a marked functional recovery (BBB 2 points; Grid-walk 30% less errors compared to control). This recovery was accompanied by an 18% increase in tissue sparing at the lesion epicentre. No gross histological changes in glial scarring were apparent. Our data demonstrate beneficial effects of an anti-CD81 antibody on functional recovery in spinal cord injured rats and suggest that this effect is mediated through a reduction in secondary tissue loss.

Reisolation of Ruminobacter parvum.

A cellulolytic gram-negative ovoid motile rod (strain GS III) was isolated from an in vitro incubation of ground barley straw in rumen fluid. The anaerobic, non-sporulating, mesophilic organism strongly answered the original description of Ruminobacter parvum (Kaars Sijpesteijn, 1948). The strain GS III fermented pyruvate, D-arabinose, D-xylose, cellobiose, sucrose, maltose, cellulose, dextrin, xylan and pectin. Products from cellobiose were D-lactate, ethanol, acetate, hydrogen and carbon dioxide. A heat-resistant factor in yeast extract that was not a B-vitamin, a metal or one of the volatile fatty acids was required for growth. The mol % G + C was 51.9. The organism was lost after prolonged subculture.

Methanogenic fermentation of benzoate in an enrichment culture.

Enrichment cultures inoculated with black mud fermented benzoate according to the stoichiometric equation: 4 C6H5CO2H+18 H2O → 15 CH4+13 CO2.Trans-2-hydroxycyclohexanecarboxylate, 2-oxo-cyclohexanecarboxylate, pimelate, caproate, butyrate, acetate, and molecular hydrogen were shown to be regular components of the culture fluid occurring in low concentrations. Inhibition of methanogenesis by chloroform, 4-chlorobutyrate, or 2-bromooctanoate resulted in a cessation of the benzoate breakdown after all intermediates had accumulated. It is proposed that benzoate is fermented via a direct reductive pathway to butyrate, acetate, H2, and CO2, whereafter butyrate is converted to acetate and H2, and the latter substrates are fermented to CH4 and CO2 by methane producers.

Some characteristics of Anaerovibrio lipolytica a rumen lipolytic organism.

Strains of Anaerovibrio lipolytica isolated from sheep- and cow-rumen contents on a linseed oil -- rumen fluid -- agar medium fermented ribose, glycerol and DL-lactate. Fermentation products from glycerol were propionate and succinate, while ribose, fructose and DL-lactate were fermented mainly to acetate, propionate and carbon dioxide. Propionate is formed in this organism by the dicarboxylic acid pathway similarly as in propionibacteria. Measurements of the rate of lipolysis by pure cultures suggest that the organism may play an important role in the lipolytic activity of rumen contents of sheep. The demonstrated fact that the cell-free lipase excreted in the culture medium can easily be adsorbed on particulate matter in autoclaved rumen fluid may explain the absence of free lipase in clarified rumen liquor.