Ronald S. Markowitz

Models of spinal cord injury: Part 3. Dynamic load technique.

Having previously studied a static load model of cord injury in rats, we report here an evaluation of a dynamic (weight drop) technique. Under general anesthesia, Sprague-Dawley rats were subjected to a laminectomy at T12, after which a 10-g weight was dropped onto a force transducer and impounder resting on the spinal cord; the weight drop distances varied in different groups from 0 (control) in increments of 2.5 cm to a maximal height of 17.5 cm. A strain gauge attached to the force transducer yielded an oscilloscopic wave form from which force of impact (peak force and impulse) was calculated. Eighty-six animals were used in this parametric study. The animals were observed for 4 weeks postinjury with two tests of motor recovery (Tarlov score for locomotion and the inclined plane test). After sacrifice at 4 weeks, the spinal cords were removed and, with the use of preset criteria, qualitative histopathological scoring of the extent of tissue damage was carried out. We found that the variable height of weight drop was capable of producing a graded injury that correlated with the force of injury (as measured by the force transducer) and with the outcome parameters of functional recovery and degree of morphological damage in the spinal cord. Histopathologically, there was a tendency to central cavitation of the cord. Both the static load and the dynamic load techniques seem to be valid models of spinal cord injury. Pathologically, however, the tissue damage after static load injury involved primarily the dorsal half of the cord. By contrast, the dynamic load technique produced central cavitation comparable to that observed in human spinal cord injury. In this respect, the dynamic model seems to be superior and its use is therefore recommended for studies of therapeutic intervention for spinal cord injury.

Test for chemotherapeutic sensitivity of cerebral gliomas: use of colorimetric MTT assay.

SummaryThis study was undertaken to evaluate the colorimetric MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide] assay as a means of testing the sensitivity of gliomas to chemotherapeutic agents in vitro. Eight human glioma established cell lines were plated in 96-well tissue culture plates and incubated for 4 days with 10 different anti-cancer agents; 5 different concentrations of each drug were tested. The MTT dye was then added to the wells, and the resulting formazan precipitate was solubilized with dimethylsulfoxide (DMSO). The spectrophotometric absorbance (measured at 570 nm) of control and experimental wells was used to calculate the cytotoxicity index (CI). Values with a CI greater than 50% growth inhibition indicated cytotoxic efficacy (sensitivity to the chemotherapeutic drug).Six of the seven (85.7%) glioma cell lines were highly sensitive at varying concentrations to mitomycin C, cisplatin, and doxorubicin. Four of the seven (57.1%) cell lines demonstrated intermediate sensitivity to mitoxantrone and vinblastine. Five of the seven (71.4%) cell lines exhibited resistance to etoposide, bleomycin, cosmegen, and BCNU. One of the cell lines tested, U-138MG, failed to produce the MTT formazan precipitate, so that the sensitivity of this cell line to the panel chemotherapeutic drugs could not be determined. The variability of the results indicates the need for an in vitro screening method to evaluate the effectiveness of clinical and experimental chemotherapeutic agents. The MTT assay provides a rapid method of screening antineoplastic agents against gliomas for cytotoxicity.

Sensorimotor-related discharge of simultaneously recorded, single neurons in the dorsal raphe nucleus of the awake, unrestrained rat.

Multi-channel, multi-neuron recording procedures were used to monitor simultaneously the spike train activity of single neurons (n=7-16 cells/animal) in the dorsal raphe (DR) nucleus of the awake, freely moving rat. Putative serotonergic and non-serotonergic neurons were distinguished from one another on the basis of established criteria, i.e. waveform shape and duration, firing pattern and firing frequency. As a group, presumed serotonergic neurons exhibited low tonic discharge rates, depressed firing after serotonin (5HT)-1a agonist administration, and, except for the transition from sleep to waking, a general insensitivity to specific sensory or motor events. By contrast, non-serotonergic cells in midline and lateral wing sub-regions of the nucleus displayed responses to a variety of sensorimotor events including locomotion, grooming, head movement, chewing, auditory stimuli, and whisker movement (both passive and active). However, within this latter group, the sensorimotor response repertoire of individual cells was not uniform. Likewise, non-5HT cells with diverse response profiles were identified in both medial and lateral sub-regions of the nucleus. Cells categorized as non-serotonergic also had varied responses to 5HT1a agonist administration. These results emphasize the diverse input/output relationships of individual DR neurons and underscore the need for a more comprehensive analysis of such properties under waking conditions in order to obtain a better understanding of the role of the DR nucleus in brain function.

Naloxone and experimental spinal cord injury: Part 2. Megadose treatment in a dynamic load injury model

We previously reported that high dose naloxone (10 mg/kg) failed to promote recovery of motor function after a static load injury of the spinal cord in rats. In the present experiments, using the more traditional dynamic weight drop model, we tested megadose naloxone administered by the intraperitoneal route in 23 rats, including saline controls (Experiment 1). Thirty minutes after a cord injury at T-12, naloxone was given i.p. in a bolus of 100 mg/kg, followed by a continuous i.p. infusion of 50 mg/kg/hour for 23 hours. Again, no benefit was observed; this raised a question regarding naloxone and its absorption and serum levels. In another study, reported separately, we found that naloxone administered by the subcutaneous route affords higher and more sustained serum levels than by i.p. administration. Consequently, in 20 rats (Experiment 2), we repeated the protocol, using subcutaneous naloxone in a bolus dose of 150 mg/kg, followed by continuous infusion of 75 mg/kg/hour for 23 hours; the result was again negative. Morphometric determination of the residual (normal) cross sectional areas of gray and white matter at the epicenter of the cord lesion showed no statistically significant difference between treated and control animals in either Experiment 1 or Experiment 2. In view of the negative findings at high dose (10 mg/kg) and megadose naloxone, it seems that a reasonable next step would be an evaluation of lower doses using a factorial research design, incorporating a range of doses of naloxone in relation to a variety of intensities of cord injury. This question will be addressed in future experiments.

Naloxone and experimental spinal cord injury: Part 1. High dose administration in a static load compression model.

Previous studies by other investigators using a dynamic weight drop injury model in cats or rats have demonstrated a beneficial effect of naloxone in promoting motor recovery after experimental spinal cord injury. The effective doses ranged from 0.8 mg (total dose) in rats to a high dose of 10 mg/kg in cats. We report here an evaluation of high dose naloxone (10 mg/kg) in a model of cord injury in rats using a static load compression technique. After induction of injury at T-12, naloxone (10 mg/kg) was administered by the intraperitoneal route, followed by five additional bolus injections over the course of the next 2 days. Animals were randomly assigned to this treatment regimen (n = 10) or to a saline control group (n = 10). The animals were observed for 4 weeks, with testing of recovery of hind limb motor function (Tarlov score and on an inclined plane). Although there were slight differences in recovery, the overall evaluation showed no statistically significant difference between the naloxone-treated and control groups. The spinal cords of the sacrificed animals were studied morphometrically; there was no statistically significant difference between the residual gray and white matter at the site of cord injury between the treated and control groups. Naloxone did not seem to promote recovery of motor function in this model of spinal cord injury.

Postmortem magnetic resonance imaging of experimental spinal cord injury: magnetic resonance findings versus in vivo functional deficit.

The relationship between the severity of the posttraumatic functional deficit and findings on magnetic resonance imaging (MRI) was investigated in a rat model of experimental spinal cord trauma. Thirty Sprague-Dawley rats were subjected to an identical, moderate, contusion injury of the spinal cord. Control animals underwent laminectomy without cord injury. The severity of the functional deficit was assessed with the Combined Behavioral Score (CBS). Animals were killed at 3, 7, 14, 21, or 28 days after injury, and the fixed, excised spinal cords were studied with MRI at 1.9 T. The lesion length was measured on sagittal spin-echo MRI. The lesion length measured on MRI was highly correlated with the CBS functional score (r = 0.56, P = 0.002). There were significant correlations between lesion length as determined by MRI and by histological morphometry (r = 0.44, P = 0.02), between histological morphometric lesion length and CBS functional deficit (r = 0.76, P < 0.001), and between the area of residual white matter at the lesion epicenter, determined by histological techniques, and the severity of functional deficit (r = -0.59, P = 0.001). A qualitative estimate of the area of preserved white matter, derived from MRI, was significantly correlated with the severity of functional deficit (r = -0.56, P = 0.006). A multiple regression of MRI-determined lesion length and MRI estimate of residual white matter versus CBS explained more than 42% of the variability of the functional deficit among these animals subjected to the same weight drop injury. We conclude that MRI parameters are reliable predictors of the severity of neurological deficit in experimental spinal cord trauma.

Experimental spinal cord injury: Mr correlation to intensity of injury

Objective This study was conducted to determine the association between lesion length measured on MRI and the severity of mechanical injury in a rat model of spinal cord trauma. Materials and Methods Sprague-Dawley rats were subjected to a modified Allen weight drop injury and killed 4 h after injury. Their fixed, excised cords were studied with MRI at 1.9 T. Results There were strong correlations between lesion length on MR images and weight drop height and square root of drop height (r2 = 0.55, p < 0.0001 and r2 = 0.63, p < 0.0001, respectively). The lesion length differences were significant versus controls for all drop heights at p values of = ≤0.002 and for the 2.5 cm animals versus the 5 and 15 cm animals (p = 0.01 and p = 0.0002, respectively), but the lengths of the 5 vs. 15 cm groups only approached significance (p = 0.06). Conclusion These results suggest that lesion length determined on MR images is a reliable indicator of the severity of trauma among animals subjected to weight drop injury.

Postmortem Magnetic Resonance Imaging of Experimental Spinal Cord Injury

The relationship between the severity of the posttraumatic functional deficit and findings on magnetic resonance imaging (MRI) was investigated in a rat model of experimental spinal cord trauma. Thirty Sprague-Dawley rats were subjected to an identical, moderate, contusion injury of the spinal cord. Control animals underwent laminectomy without cord injury. The severity of the functional deficit was assessed with the Combined Behavioral Score (CBS). Animals were killed at 3, 7, 14, 21, or 28 days after injury, and the fixed, excised spinal cords were studied with MRI at 1.9 T. The lesion length was measured on sagittal spin-echo MRI. The lesion length measured on MRI was highly correlated with the CBS functional score (r = 0.56, P = 0.002). There were significant correlations between lesion length as determined by MRI and by histological morphometry (r = 0.44, P = 0.02), between histological morphometric lesion length and CBS functional deficit (r = 0.76, P < 0.001), and between the area of residual white matter at the lesion epicenter, determined by histological techniques, and the severity of functional deficit (r = -0.59, P = 0.001). A qualitative estimate of the area of preserved white matter, derived from MRI, was significantly correlated with the severity of functional deficit (r = -0.56, P = 0.006). A multiple regression of MRI-determined lesion length and MRI estimate of residual white matter versus CBS explained more than 42% of the variability of the functional deficit among these animals subjected to the same weight drop injury. We conclude that MRI parameters are reliable predictors of the severity of neurological deficit in experimental spinal cord trauma.

Spinal cord injury in the monkey: rate of cord cooling and temperature gradient during local hypothermia

We have investigated the effectiveness of surface cord cooling in reducing spinal cord temperature with respect to the rate of cooling and the temperature gradient within the spinal cord parenchyma. Another question of some practical importance if spinal cooling is to be used clinically is that of the temperature within the spinal cord as a function of the dura being intact or open. In five monkeys, a laminectomy was carried out at T-10 and an impact injury of 350 g-cm force was applied to the spinal cord with the dura intact. Hypothermic perfusion with Elliotts B solution (artificial cerebrospinal fluid) was started, and we measured temperatures in the spinal cord with a thermistor probe mounted on a stereotactic drive. A series of measurements were made with the dura intact and the measurements were then repeated with the dura open. For comparison, we also recorded temperatures in one animal that had not been cord-injured. The rate of cord cooling was rapid during the first 3 minutes of hypothermic perfusion, after which there was a slight further reduction in cord temperature; a low level plateau of 6.7 degrees C was reached within 21 minutes after the start of cooling. The temperature gradient at varying depths of the spinal cord was approximately 6 degrees C (3.8 degrees C at the posterior surface to 10.1 degrees C in the deepest portion of the spinal cord), representing a temperature gradient of 1.3 degrees C/mm of cord tissue. Transmission of the cooling effect from perfusate to the spinal cord was not appreciably affected by the dura being intact or open. (Neurosurgery, 5: 583--587, 1979).

Feasibility of intracranial surgery in the rat fetus: model and surgical principles.

The feasibility of intracranial surgery in the rat fetus is demonstrated by the use of a microsurgical model. In utero craniotomy and cerebral incision have been performed successfully without compromise of the fetal-maternal unit. Maternal and fetal survival rates of 95 and 93%, respectively, are reported. Several principles of fetal surgery have been established and are discussed. These include the inhibition of uterine contraction, monitoring of physiological variables, timing of operation, microsurgical technique, and preservation of amniotic fluid. The fetal rat model is amenable to antenatal research because it fulfills certain criteria, including accurately timed pregnancies, littermate controls, inexpensiveness, and animal availability.