Spinal shock

Background & objectives: In vitro models of anoxia have revealed severe changes in neuronal functions after ischaemia but not after aglycaemia, although hypoglycaemia produced severe neuronal dysfunctions sometimes leading to coma. The present study was therefore undertaken to examine and compare the effects of aglycaemia with that of ischaemia on synaptic transmission in vitro.

Methods: Spinal cord from the neonatal rat was isolated, hemisected and placed in a chamber perfused with standard physiological solution. The stimulation of a dorsal root elicited monosynaptic (MSR) and polysynaptic (PSR) reflex potentials in the segmental ventral root. The effects of suprefusing glucose free medium (aglycaemia) and superfusing glucose free and O2 free medium (ischaemia) were examined on these reflexes.

Results: Superfusion of aglycaemic solution did not alter the magnitude of MSR or PSR in the first 15 min and subsequently there was a time-dependent depression of the reflexes (P

Interpretation & conclusion: The results of the present study indicate that the aglycaemia and ischaemia depressed the synaptic transmission to the same extent though there were differences in their onset and progress. Aglycaemia involves N-methyl-D-aspartate (NMDA) receptor-dependent (Mg^sup 2+^ sensitive) mechanism, while ischaemia-induced depression involves other mechanisms in addition to NMDA.

Key words Anoxia - hypoxia - monosynaptic reflex - N-methyl-D-aspartate receptor - polysynaptic reflex

The anoxia is a condition of decreased oxygen (O2) at the cellular level and has been implicated in the pathogenesis of neurodegenerative diseases1,2. The anoxia can result from the lack of O2 (hypoxia), the lack of substrate (aglycaemia) or their combination (ischaemia). Whatever may be the cause of anoxia, there is cellular ATP deficiency in these conditions. The ATP depletion and neuronal changes can also be seen in the in vitro models of hypoglycaemia (glucose free) or ischaemia (glucose free and O2 free)3. Besides ATP depletion, depolarization of neuronal membrane is also reported3. The depolarization was attributed to the excitatory amino acids (glutamate, aspartate and taurine) released by the anoxic insults. The depletion of ATP, the magnitude of depolarization and the quantity of transmitters released are much greater in ischaemia than in other conditions3. Further in ischaemia, additional transmitters such as norepinephrine, dopamine and 5-hydroxytryptamine (5-HT) are also released3-7. The glutamate or other agonists so released from the neuronal cells in turn elevate the intracellular Ca^sup 2+^ leading to the neuronal damage8,9. However in a study10 hypoglycaemia produced no cellular damage as seen with ischaemia, though the hypoglycaemic coma is a known entity. Thus, discrepancies are reported in the actions of different types of anoxic conditions on the neuronal functions. The present study was therefore, undertaken to examine the effects of aglycaemia and ischaemia on the synaptic transmission, which is an indicator of the functional activity of neurons. The synaptic transmission evoked at ventral root by dorsal root stimulation and the responses primarily involved Ia-[alpha] motoneuron synapse. This is having monosynaptic and polysynaptic components11-15 and involves glutamatergic transmission16,17. Since glutamate is involved in ischaemic insults3,7,9,10, we used a synapse where glutamate is a primary transmitter.

Material & Methods

Animals, anaesthesia and dissection: The experiments were performed in the Department of Physiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi after obtaining clearance from the ethics committee of the Institute for conducting animal experimentation. The methods for the dissection and recording procedures were used as described earlier11-13. Under the ether anaesthesia, the spinal cord was dissected from the 4-6 day old rats (Charles-Foster strain) of either sex. The spinal cord was dissected out carefully from the vertebral column, hemisected sagitally, and transferred to a small Plexiglass bath (volume

Stimulation and recording: The suction electrodes were prepared by fire polishing the borosilicate glass capillary tubes (WPI, USA). The corresponding segmental dorsal and ventral roots were sucked gently into the glass capillary electrodes with reduced tip size to fit the roots tightly by fire polishing. The stimulation of the dorsal root with rectangular pulses of 0.5 msec duration and supramaximal voltage at 0.1 Hz (obtained from a stimulator from Biodevices, Ambala, India) evoked reflex potentials in the corresponding segmental ventral root. These reflex potentials were amplified (AC/DC preamplifier from Harvard Instruments, UK), monitored on an oscilloscope (BPL, India), digitized and stored in a personal computer using A-D card (PCL-208 from Dynalog Microsystems Ltd., Mumbai, India). In our experiments after attaching the roots with electrodes, the reflexes were monitored at 15 min intervals till the reflexes in two consecutive or successive recordings remained the same which usually occurred between 1-3 h. This time was required for recovery of spinal shock, and sealing of the roots to the electrodes to form high resistance bridges.

Experimental protocol: After stabilization (1-3 h), the averaged signal of 5-6 successive reflex potentials was recorded. The aglycaemia was produced by superfusing glucose free physiological solution bubbled with 100 per cent O2, the ischaemia was produced by superfusing glucose free physiological solution without O2, bubbling and the hypoxia by superfusing standard physiological solution without O2 bubbling. In a typical experiment after recording the initial reflex response, the cord was superfused with either aglycaemic or ischaemic solution till the abolition of the reflex (n = 6 for each of the conditions). The averaged signal was recorded initially and then at every 5 min interval in case of Mg^sup 2+^ free medium and 15 min interval for the experiments in the presence of Mg^sup 2+^ medium. The recordings were continued till the abolition of reflexes (within 40 min) or up to 80 min. At the end, the cords were washed with the physiological solution bubbled with O2 for 30 min. In separate groups, the aglycaemia (n = 6) or the ischaemia (n = 6) was produced by superfusing the physiological solution containing Mg^sup 2+^ (1.3 mM) and the recordings were made up to 80 min (twice the time required for abolition of reflexes in Mg^sup 2+^ free conditions). Control experiments (n = 4) were conducted to know the time related effect on MSR and PSR without exposing the cords to any of the hypoxic conditions for 80 min.

Data analysis: The area under the reflex curve was computed at different time intervals and was expressed as per cent of the initial. The mean ± standard error (SE) were computed from the pooled data. The time response relationship was compared using one way ANOVA. The difference between two points was tested by using Student-Newman-Keuls test. The differences between the corresponding points in the absence and the presence of Mg^sup 2+^ in aglycaemia or ischaemia were compared by two way ANOVA and the comparisons between the two corresponding points were performed by using Student-Newman-Keuls test; P

Results & Discussion

More than two reflex potentials were recorded from the ventral root after stimulating the corresponding dorsal root (Fig. 1 control). The latency of the first potential was around 5 msec and was considered as monosynaptic reflex (MSR). The latency of the second potential was around 12 msec and was considered as polysynaptic reflex (PSR). These latencies for MSR and PSR are similar to our earlier reports12-14. The reflexes in control situation (without any hypoxic condition) remained unaltered during the period of 80 min.

Superfusion of aglycaemic solution did not alter the magnitude of MSR and PSR in the first 15 and 10 min respectively and subsequently there was a time-dependent depression of the reflexes (P

The superfusion of ischaemic solution produced a progressive decline in the MSR or the PSR in a time-dependent manner starting from the very beginning (P

In a separate series, the experiments were conducted to examine the effect of hypoxia alone on synaptic transmission by superfusing the cords with a standard physiological solution without O2 bubbling. At 5 min, a significant depression of reflexes (6-12%) was observed (P

Superfusion of Mg^sup 2+^ (1.3 m M) containing solution abolished the PSR and decreased the MSR by 30 per cent. These effects of Mg^sup 2+^ on MSR and PSR are consistent with our earlier observations12,14. The aglycaemia-induced depression of MSR was blocked in the presence of Mg^sup 2+^ (P

In the presence of Mg^sup 2+^, the ischaemia-induced depression was attenuated significantly (P

The present results demonstrate that the aglycaemia and ischaemia depressed the synaptic transmission to the same extent although there were differences in their onset and progress. Presence of Mg^sup 2+^ prevented the aglycaemia-induced depression completely and the ischaemia-induced depression partially.

The initial resistance observed in aglycaemia may be due to the presence of cellular energy substrates available for the oxidative metabolism. The energy is required for Na^sup +^-K^sup +^ ATPase pump, for the maintenance of the membrane polarity, transmitter release and other cellular functions. The experiment with hypoxia shows the absence of initial resistance as observed in aglycaemia. Further, the initial depression of the reflexes was exaggerated to a greater extent in ischaemia as compared to hypoxia and may be correlated to the greater decrease in ATP levels3. The observations with hypoxia also rule out the possibility of lack of O2 alone for the ischaemia-induced depression. However, the initial resistance observed in aglycaemia (where O2 was freely available but not glucose) indicates the involvement of oxidative metabolism.

The PSR and a fraction of MSR have been shown to involve the N-methyl-D-aspartate (NMDA) receptors, while the remaining part of MSR is mediated through non-NMDA mechanism12,14,15. The decrease in the magnitude of MSR and the abolition of PSR completely in the presence of Mg^sup 2+^ observed in this study, support the involvement of NMDA in PSR as shown earlier12,14. Thus, the complete protection offered by Mg^sup 2+^ in case of aglycaemia indicate that the aglycaemia-induced depression is solely mediated through the NMDAdependent mechanism. In case of ischaemia, the Mg^sup 2+^ prevented the depression for a period, after that there was a drastic decline to abolish the reflex at 80 min. The initial period of protection during ischaemia in the presence of Mg^sup 2+^ may involve NMDA mediated mechanisms and the subsequent depression even in the presence of Mg^sup 2+^ indicate the mechanisms other than NMDA. In the spinal cord, NMDA or its analogs exert excitatory actions16,17. We have earlier shown12 that the NMDA depressed the MSR and PSR in isolated spinal cord preparation. It was suggested that the depression induced by NMDA might be due to the stimulation of inhibitory transmission or due to the depolarization block12. Similar mechanisms may be responsible for the aglycaemia or ischaemia-induced depression of the reflexes.

In a study10 elsewhere, hypoglycaemia failed to produce the neuronal damage but ischaemia produced severe damage. If the neuronal damage is the cause for the synaptic depression, the aglycaemia should not produce the depression. In the present study the abolition time of synaptic depression by aglycaemia was found to be equivalent to that of ischaemia. Therefore, the factors other than neuronal damage seem to be involved for the synaptic depression.

In summary, ischaemia and aglycaemia abolished the spinal synaptic transmission simultaneously involving different mechanisms. The aglycaemia-induced depression involved mainly NMDA dependent (Mg^sup 2+^ -sensitive) mechanism while the ischaemia-induced depression involved other mechanisms besides NMDA.

Acknowledgment

The financial support received from the Defence Institute of Physiology and Allied Sciences (DIPAS), Defence Research & Development Organization (DRDO), Delhi is acknowledged. The first author is a recipient of fellowship from the Indian Council of Medical Research, New Delhi, India.

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Archana Jha, S. Dasgupta & S.B. Deshpande

Department of Physiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India

Received March 25, 2003

Reprint requests : Dr S.B. Deshpande, Professor, Department of Physiology, Institute of Medical Sciences Banaras Hindu University, Varanasi 221005, India

e-mail : desh48@yahoo.com

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