Rat Spinal Subarachnoid Space Research Articles

An improved method for protecting and fixing the lumbar catheters placed in the spinal subarachnoid space of rats

The chronic intrathecal catheterization of the subarachnoid space in the rat is a very useful model in the research of neuroscience, anesthesiology, neurotoxicity, and etc. To avoid high postoperative mortality and neurological impairment, the catheterization from the lumbar interspace has been widely used in recent years. But one of the most discouraging disadvantages is that the external portion of the chronic indwelling catheters is easily damaged by rats. Besides, the short length of catheter left in the subarachnoid space in addition to its slimness makes it difficult for proper fixation. To improve the abovementioned situations, we designed a protector to protect the external portion of the catheters and developed a simple method for better fixation. Results suggest that this is an excellent method for protecting and fixing the lumbar catheters placed in the subarachnoid space of rats, and this method did not affect the sensory and motor function of the rat. The general conditions of the rats were unswayed.

The significance of intrathecal catheter location in rats.

Although chronic intrathecal catheterization is a widely used method in rats, few calibration experiments have been performed. In this study, we investigated the correlation between the side position of the catheter tip and the side differences observed in the motor and sensory disturbances after intrathecal administration of lidocaine to a large number of rats. The existence of a sensory block was determined by the paw withdrawal test. The motor impairment was assessed by observing the complete clubbing of the hindpaw and measuring the hindpaw grip strength. After experimental use, we established the position of the catheter tip. The catheter tips were variously located in all directions of the transverse plane in the rat spinal subarachnoid space. Lidocaine administration (100 or 500 microg/5 microL; n = 264 and 112, respectively) led to dose-dependent motor and sensory disturbances. The effect of 100 microg of lidocaine exhibited side differences; i.e., the extents of both motor (r = 0.77) and sensory (r = 0.60 and r = 0.67 for the right and the left side, respectively) disturbances correlated significantly with the location of the catheter tip. Our data have shown that detection of the paralytic and/or antinociceptive effect of small-dose lidocaine before planned experiments is a simple and reliable method for prediction of the location of the catheter tip. We suggest that the position of the catheter might cause side differences in the drug effect, especially if small doses of drugs are administered and their effects are investigated on both sides.

The Significance of Intrathecal Catheter Location in Rats

Although chronic intrathecal catheterization is a widely used method in rats, few calibration experiments have been performed. In this study, we investigated the correlation between the side position of the catheter tip and the side differences observed in the motor and sensory disturbances after intrathecal administration of lidocaine to a large number of rats. The existence of a sensory block was determined by the paw withdrawal test. The motor impairment was assessed by observing the complete clubbing of the hindpaw and measuring the hindpaw grip strength. After experimental use, we established the position of the catheter tip. The catheter tips were variously located in all directions of the transverse plane in the rat spinal subarachnoid space. Lidocaine administration (100 or 500 μg/5 μL;n = 264 and 112, respectively) led to dose-dependent motor and sensory disturbances. The effect of 100 μg of lidocaine exhibited side differences; i.e., the extents of both motor (r = 0.77) and sensory (r = 0.60 and r = 0.67 for the right and the left side, respectively) disturbances correlated significantly with the location of the catheter tip. Our data have shown that detection of the paralytic and/or antinociceptive effect of small-dose lidocaine before planned experiments is a simple and reliable method for prediction of the location of the catheter tip. We suggest that the position of the catheter might cause side differences in the drug effect, especially if small doses of drugs are administered and their effects are investigated on both sides.

Distribution of intrathecal catheter positions in rat.

Although drug administration through an intrathecal catheter is widely used in the study of spinal pharmacology, the catheter positions in transverse plane that may cause a limited spread of a solution remain unclear. In the first step to clarify this issue, the distribution of the intrathecal catheter position was investigated in rats. A polyethylene catheter (PE-10) was inserted intrathecally 8.5 cm through the atlanto-occipital membrane, aiming the tip of the catheter to the dorsal surface of the spinal cord. Three or four weeks after the implantation of the catheter, 83 rats were transcardiacally perfused with fixative solution. The catheter positions were investigated longitudinally and transversely by cutting the spinal cord segmentally through the intervertebral disk at different spinal levels. Seventeen rats were excluded from further data analysis. Transversely, catheters were located in the left lateral subarachnoid space in 23 rats (35%), in the right lateral in 15 (23%), in the dorsal in 22 (33%), and in the ventral in 6 (9%). Longitudinally, catheter position was significantly higher in the ventral group (median, T9) than that in the dorsal group (T11/12) (P<0.01). Chronic intrathecal catheters were variously located in the rat spinal subarachnoid space in the transverse plane, and lateral subarachnoid placement of the catheter (58%) was frequently observed, whereas dorsal subarachnoid placement occurred in 33%.

Chapter 26 The alleviation of pain by cell transplantation

Publisher Summary The mushrooming of the field of cellular transplantation in recent years has given rise to a new way of supplying analgesic molecules. In animal models, cell-transplant analgesia has proven successful both with cells from primary tissue (mostly in studies with adrenal medullary chromaffin cells) and with immortalized, bioengineered cell lines that secrete pain-reducing neuroactive substances. The choice of donor material for cell-transplant analgesia presents some interesting contrasts with other neural transplant therapies. Homotypic tissue is a realistic choice as replacement material for repair of deficits, such as those seen in Parkinson's disease or spinal trauma. However, heterotypic tissue shows an apparently coincidental but useful concordance with the function of the local host tissue. This is most remarkable in the case of the adrenal medulla for spinal analgesia. Preclinical pain models of several types have by now confirmed that adrenal medullaty chromaffin cells can be analgesic when transplanted into the rat's spinal subarachnoid space.

A method for increasing the viability of the external portion of lumbar catheters placed in the spinal subarachnoid space of rats

A method for direct catheterization of the lumbar subarachnoid space has recently been developed by Storkson et al. (1996) (J Neurosci Methods 1996;65:167–172) that may potentially improve upon the widely used method of Yaksh and Rudy (1976) (Physiol Behav 1976;17:1031–1036). This ‘catheter-through-a-needle’ technique inserts the catheter between lumbar vertebrae 5 (L5) and 6 (L6), which has been shown to reduce neurological impairment and post-surgical deaths. However, employing this technique allows the external portion of the chronic indwelling catheters to be easily damaged, resulting in ≈50% attrition within 4 days after surgery. Therefore, we developed an easy and inexpensive method for protecting the external portion of the catheter that enhances catheter viability beyond 14 days after catheter implantation while also maintaining a low injection volume (8 μl). Moreover, this modification does not significantly alter the implantation methods developed by Storkson et al. (1996) (J Neurosci Methods 1996;65:167–172) and allows for more optimal catheter materials to be incorporated. Chronically implanted catheters ( n=70) with the external portion of the catheter protected, resulted in 4% attrition 7 days after surgery and 11% attrition 14 days after surgery. Approximately 5.5% of animals implanted showed very mild and transient neurological impairment.

Antinociception produced by the peptidase inhibitor, RB 101, in rats with adrenal medullary transplant into the spinal cord

This study was undertaken to investigate the effects induced by the systemic administration of RB 101 [ N-{( R, S)-2-benzyl-3[( S)(2-amino-4-methylthio)butyl dithio]-1-oxoprpyl}- l-phenylalanine benzyl ester], a mixed inhibitor of the enkephalin catabolism able to cross the blood–brain barrier, in antinociception produced by adrenal medullary tissue transplanted in the rat spinal subarachnoid space. For this purpose, the antinociceptive responses induced by intravenous (i.v.) administration of RB 101 were evaluated in the tail-flick in rats transplanted 28 and 56 days before the test. Systemic administration of RB 101 induced antinociceptive effects in sham-operated rats, as previously reported. RB 101 also enhanced significantly the antinociception produced by the autotransplant 28 and 56 days after surgery. The antinociceptive responses of RB 101 in both sham-operated and autotransplanted rats were blocked by naloxone, but were not modified by the noradrenergic antagonist, phentolamine, suggesting a selective involvement of opioid mechanisms. The present results indicate that the inhibitors of enkephalin catabolism enhance the antinociception induced by adrenal medullary transplants.

AMPA receptor-mediated slow neuronal death in the rat spinal cord induced by long-term blockade of glutamate transporters with THA

Excitotoxicity secondary to the loss of glutamate transporters (GluT) has been proposed as a possible pathogenetic mechanism for neuronal degeneration in amyotrophic lateral sclerosis. We therefore investigated whether prolonged in vivo pharmacologic inhibition of GluT would result in neuronal damage in the rat. dl-Threo- β-hydroxyaspartate (THA), a potent GluT inhibitor, and glutamate were continuously infused into the rat spinal subarachnoid space by using a mini-osmotic pump. Animals that received both THA and glutamate, but not those received either singly, displayed tail paralysis with or without hind-limb paralysis and urinary incontinence after the third postoperative day. Pathologically, symptomatic animals exhibited neuronal loss with a variable extent of gliosis preferentially involving the dorsal horn of the lumbosacral cord. In the rostral spinal segments adjacent to those regions of intense pathologic changes, small neurons in the dorsal horn were selectively destroyed, a pattern similar to the late-onset neuronal damage induced by continuous intrathecal administration of 1-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) [R. Nakamura et al., Brain Res. 654 (1994) 279–285]. These behavioral and pathologic changes were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), suggesting that pharmacologic blockade of GluT causes selective neuronal damage in vivo by AMPA receptor activation.

Acute and late neurotoxicity in the rat spinal cord in vivo induced by glutamate receptor agonists

We investigated glutamate receptor-mediated neurotoxicity in vivo by means of infusing three specific agonists for non-NMDA receptors (acromelic acid A (ACRO), kainic acid and 1-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)) into the adult rat spinal subarachnoid space. ACRO induced long-lasting pure motor, rigid-spastic paraparesis in a dose-dependent manner (EC 50: 220 pmol/h) that was accompanied by selective degeneration of spinal interneurons; leaving large anterior horn cells intact. Kainate and AMPA induced paraplegia but with relatively non-selective neuronal damage when given in doses more than 40-fold larger than those required for ACRO. When AMPA (> 100 nmol/h) was infused continuously using a mini-osmotic pump for more than 2 days, rats displayed progressive changes in motor behavior due to extensive damage in the caudal spinal cord where small neurons in the dorsal horns were the most vulnerable. Co-administration of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) specifically prevented neurotoxicity, suggesting a non-NMDA receptor-mediated mechanism. These results indicate that the non-NMDA receptor is heterogeneous, mediating neuronal damage with different selectivity. It is also suggested that chronic activation of glutamate receptors is capable of inducing slowly progressive neuronal death, which suggests relevance to the pathogenesis of ALS.

Effect of Amitriptyline on the Analgesia Induced by Adrenal Medullary Tissue Transplanted in the Rat Spinal Subarachnoid Space as Measured by an Experimental Model of Acute Pain

The effect of short and long term amitriptyline (AMI) treatment on the analgesia induced by adrenal medullary autotransplant into the subarachnoid space was investigated in rats. For this purpose, two experiments were carried out. In the first one, the rats were chronically treated for 28 days after transplantation. In the second experiment, rats were treated for 28 days starting 28 days after surgery. Before starting the experiments, basal levels were tested. Tail-flick latencies were checked at Day 4 and Day 28 in the first experiment and at Day 28 and Day 56 in the second. AMI itself did not induce any tail-flick modification after 28 or 56 days. However, it increased the transplantation-induced analgesia at Day 28 in the first experiment and at Day 56 in the second. These results are interpreted in the sense of the ability of AMI to enhance the effects of both monoamine and opioids previously released by the transplantation.

Alleviation of neuropathic pain symptoms by xenogeneic chromaffin cell grafts in the spinal subarachnoid space

Recent data have suggested that adrenal medullary tissue allografts in the spinal cord subarachnoid space, by releasing catecholamines and opioid peptides, attenuate responses to various acute noxious stimuli and chronic pain-related behaviors. However, the application of this approach is limited by the low availability of allogeneic donor material. Alternatively, chromaffin cells from xenogeneic sources such as the bovine adrenal medulla are plentiful and simple to extract. The goal of this study was to evaluate the potential for bovine chromaffin cell xenografts in the rat spinal subarachnoid space to alleviate chronic pain. This was assessed in an animal model of neuropathic pain induced by loose ligation of the sciatic nerve, which resulted in allodynia, hyperalgesia, and skin temperature abnormalities. Two weeks after nerve injury, animals were implanted with either isolated bovine chromaffin cells or control bovine adrenal fibroblasts in the spinal subarachnoid space at the level of lumbar enlargement and immunosuppressed with cyclosporine A. In animals with chromaffin cell implants, but not fibroblast implants, both cold allodynia and thermal hyperalgesia were markedly reduced or eliminated as early as 1 week following implantation and hind paw skin temperature asymmetry was also normalized. These beneficial effects were maintained without decrement or apparent tolerance for the 9 week course of the symptomology. The analgesic effects of chromaffin cell grafts were partially attenuated following i.t. injection of naloxone and phentolamine separately and in combination, suggesting involvement of spinal opioid and α-adrenergic receptors. Following termination of behavioral studies, immunocytochemical analysis revealed robust survival of chromaffin cells in the implants. These results demonstrate that chromaffin cell xenografts may be effective in alleviating pain of neurogenic origin.

Antinociception following implantation of mouse B16 melanoma cells in mouse and rat spinal cord

B16 F1C29 melanoma cells, which are thought to contain and release catecholamines, were implanted in mouse and rat spinal subarachnoid space. B16 F1C29 cell implants augmented the antinociceptive effect of morphine in tail-flick test, and this interaction was blocked by either the α 2-adrenergic antagonist idazoxan or the opioid antagonist naloxone. B16 F1C29 cell implants also augmented the antinociceptive effect of the catecholamine re-uptake blocker desipramine. Substance P-induced biting and scratching behaviors were inhibited in mice receiving B16 F1C29 cell implants, and this effect of B16 F1C29 cell implants was blocked by the α 2-adrenergic antagonist idazoxan. Mice receiving B16 F1C29 cell implants showed tolerance to intrathecal administration of the α 2-adrenergic agonist UK 14304. These results suggest that B16 cell implant-induced antinociception was mediated by catecholamines secreted from the cell implants and acting at spinal α 2-adrenergic receptors. Spinal implantation of catecholamine-releasing cells may provide an alternative approach for the therapy of chronic intractable pain and a useful model to study α 2-adrenergic receptor tolerance.

Transplants of immunologically isolated xenogeneic chromaffin cells provide a long-term source of pain-reducing neuroactive substances

Adrenal medullary chromaffin cells are a potential source of neuroactive substances for transplantation into the CNS to alleviate neurochemical deficits. In particular, work in our laboratory has suggested that adrenal medullary transplants in the spinal subarachnoid space can alleviate pain by providing sustained local delivery of catecholamines and opioid peptides. One of the major limitations for clinical application of neural transplantation is the availability of donor material in sufficient quantities. This limitation may be overcome by the use of xenogeneic donors if long-term graft rejection can be prevented. The purpose of this study was to assess whether xenogeneic chromaffin cells immunologically isolated by semipermeable membranes could survive and continue to reduce pain when transplanted into the CNS. Isolated bovine chromaffin cells were encapsulated by semipermeable polymer membranes and implanted into the rat spinal subarachnoid space. Pain sensitivity was assessed at several intervals up to 3 months following implantation. Results indicated that encapsulated bovine chromaffin cell implants, but not empty control capsules, could repeatedly reduce pain sensitivity with nicotine stimulation for the duration of the study. This response was dose related, indicating that pharmacologic integrity of the transplanted chromaffin cells is retained. The analgesia induced by encapsulated chromaffin cell implants could be attenuated by the opiate antagonist naloxone and the alpha-adrenergic antagonist phentolamine, suggesting the involvement of both opioid peptides and catecholamines in mediating this response. In addition, in vitro neurochemical studies of recultured capsules revealed sustained release of Met-enkephalin and catecholamines from encapsulated cells 3 months following implantation into the spinal subarachnoid space.(ABSTRACT TRUNCATED AT 250 WORDS)

Study on the Receptor Subtypes Mediating the Analgesic Action of an Enkephalin Analog, Tyr-D-Met(O)-Gly-EtPhe-NHNHCOCH3·AcOH (EK-399)

The analgesic action of the enkephalin analog EK-399 (Tyr-D-Met(O)-Gly-EtPhe-NHNHCOCH3·AcOH) and the subtypes of the opiate receptors mediating the action were studied. The analgesic effect of subcutaneously injected EK-399 was ten times as potent as that of morphine in the rat tail flick test. EK-399 had a longer latency time and duration time than morphine. The analgesic action of EK-399 injected into the rat spinal subarachnoid space was about 800 (1800 in molar ratio) times as potent as that of morphine in the hot plate test. EK-399 had high affinities for both μ and δ opiate receptors in the rat brain receptor binding assay. The apparent pA2 values with naloxone were 7.65 for morphine and 5.98 for EK-399 in the rat tail flick test; the difference was significant. A cross tolerance between EK-399 and morphine was examined in the rat tail flick test. Although morphine tolerant rats showed no tolerance to EK-399, EK-399 tolerant rats showed a clear tolerance to morphine. These results indicate that EK-399 has a potent and long lasting analgesic effect via opiate receptors in rats. In addition to μ-receptors, δ-receptors may be involved in its analgesic mechanism.

Study on the receptor subtypes mediating the analgesic action of an enkephalin analog, Tyr-D-Met(O)-Gly-EtPhe-NHNHCOCH3.AcOH (EK-399).

The analgesic action of the enkephalin analog EK-399 (Tyr-D-Met(O)-Gly-EtPhe-NHNHCOCH3.AcOH) and the subtypes of the opiate receptors mediating the action were studied. The analgesic effect of subcutaneously injected EK-399 was ten times as potent as that of morphine in the rat tail flick test. EK-399 had a longer latency time and duration time than morphine. The analgesic action of EK-399 injected into the rat spinal subarachnoid space was about 800 (1800 in molar ratio) times as potent as that of morphine in the hot plate test. EK-399 had high affinities for both mu and delta opiate receptors in the rat brain receptor binding assay. The apparent pA2 values with naloxone were 7.65 for morphine and 5.98 for EK-399 in the rat tail flick test; the difference was significant. A cross tolerance between EK-399 and morphine was examined in the rat tail flick test. Although morphine tolerant rats showed no tolerance to EK-399, EK-399 tolerant rats showed a clear tolerance to morphine. These results indicate that EK-399 has a potent and long lasting analgesic effect via opiate receptors in rats. In addition to mu-receptors, delta-receptors may be involved in its analgesic mechanism.

Intrathecal N-methyl- d-aspartate (NMDA) activates both nociceptive and antinociceptive systems

Injection of the excitatory amino acid N-methyl- d-aspartate (NMDA) into the spinal subarachnoid space of rats produces both hyperalgesic and analgesic effects. At lower concentrations (0.5 mM) little behavioral effect is elicited by the drug. However, brief hyperalgesia followed by several minutes of analgesia can be detected in these animals. Higher concentrations of the drug produce vocalization, caudally directed scratching and biting and hyper-responsiveness to light touch. The NMDA antagonist, arginine vasopressin, produces analgesia when injected by itself and completely reverses all effects of NMDA. NMDA-induced analgesia, but not hyperalgesia, is reversed by intrathecal administration of naloxone, methylsergide and phentolamine. The analgesic effects of both agonist and antagonist are markedly potentiated by spinalization. These results suggest the involvement of NMDA receptors in both the transmission of pain and the mediation of spinal segmental pain inhibitory mechanisms.

Hyperalgesia produced by the intrathecal administration of tryptamine to rats

Tryptamine was applied directly into the spinal subarachnoid space of rats via permanently indwelling cannulas. Changes in pain-perception were measured by changes in the latency of the tail-flick in response to a radiant heat source of low intensity. While an intrathecal injection of serotonin has been previously shown to be analgesic, exogenous tryptamine produced dual effects on the pain-threshold, depending on the dose of tryptamine injected. Low doses of tryptamine (100 and 200 μg/rat) injected intrathecally onto the sacral area of the spinal cord appeared to be hyperalgesic by significantly decreasing the average tail-flick latency by 5 min after injection. Administration of the serotonin antagonist methysergide alone was without effect on the average tail-flick reaction time when injected either intrathecally or subcutaneously. However, pretreatment with either methysergide or cinanserin not only failed to inhibit tryptamine's potentiation of nociception, but actually enhanced the hyperalgesia produced by tryptamine. In contrast, a dose of 400 μg of tryptamine significantly increased the average tail-flick latency, suggesting an analgesic effect at this higher dose. This analgesic effect of 400 μg of tryptamine was completely inhibited by subcutaneously administered methysergide, while intrathecally injected methysergide produced even greater decreases in the tail-flick latencies after this high dose of tryptamine. These results suggest that tryptamine, although it differs from serotonin by only one hydroxyl group, may play a role in nociception which is opposite that played by serotonin.

The thermoregulatory effects of noradrenaline, serotonin and carbachol injected into the rat spinal subarachnoid space.

1. We have examined the effects on thermoregulation in the rat of noradrenaline bitartrate (NA), 5-hydroxytryptamine hydrochloride (5-HT) and carbamylcholine chloride (CCh) injected into the lumbar spinal subarachnoid space via a chronic indwelling catheter.2. Intrathecal injections of the monoamines and CCh reproducibly affected thermoregulation, whereas injections of control solutions had no effect.3. Intrathecal injections of NA (0.01-0.30 mumol) produced a dose-dependent hypothermia associated with a decrease in tail skin vasomotor tone. Shivering activity was not depressed during the hypothermia and sometimes increased. Intrathecal administration of the alpha-adrenergic agonist clonidine (0.0175-0.070 mumol) elicited changes in T(c) and T(sk) similar to those induced by intrathecal NA.4. Intrathecal 5-HT (0.030-0.90 mumol) elicited a dose-dependent hyperthermia accompanied by increased tail skin vasomotor tone and increased shivering.5. CCh injected intrathecally (0.001-0.06 mumol) evoked a dose-dependent hyperthermia. During the period when core temperature was rising, tail skin vasomotor tone increased and shivering-like activity was present. Once the maximum core temperature had been reached, tail skin vasodilatation occurred. Vasodilatation persisted until core temperature had returned to normal.6. Intravenous injections of 5-HT (0.30 and 0.90 mumol) or CCh (0.006 and 0.03 mumol) caused no thermoregulatory effect. The effects of these agents injected intrathecally were therefore not due to an action in the periphery.7. Intravenous infusions of NA (0.06 and 0.10 mumol) produced hypothermia and transient tail skin vasodilatation. We suggest that an action at peripheral sites may have contributed to the effects produced by intrathecal injection of this monamine.8. These findings suggest that spinal noradrenergic, serotonergic and cholinergic synapses may be importantly involved in the control of body temperature in the rat. The possible functional roles of these synapses and the putative spinal sites of action of the injected substances are discussed.

An improved method for chronic catheterization of the rat spinal subarachnoid space

A method for chronic catheterization of the rat spinal subarachnoid space is described. The catheters are simple to make and the catheterization procedure circumvents many of the problems associated with earlier published technics.

Clearance of macromolecular and particulate substances from the cerebrospinal fluid system of the rat

Arachnoid villi in the intracranial dural sinuses constitute the principal sites for absorption of proteins and particulates from the cerebrospinal fluid (CSF) system. Although arachnoid villi in the rat are morphologically less complex than those found in other mammals, their resistance to CSF outflow, as assessed by a graded series of contstant flow manometric infusions, is similar to that found in other species. Moreover, inulin and polystyrene beads, when infused into the spinal subarachnoid space of rats, are rapidly cleared from the CSF system into intracranial dural sinuses. Inulin appeared in sinus blood 3 minutes after onset of infusion and reached concentrations 26 times greater than those found in the systemic circulation; particulate matter in the form of 0.5 micrometer polystyrene beads showed similar efflux characteristics. Hence, the CSF system of the rat is functionally similar to that found in other mammalian species, with arachnoid villi constituting a major efflux route for clearance of macromolecular and particulate substances.