It is well established that peripheral nerve crush injury, during early postnatal development, results in significant loss of motor neurons and extensive muscle atrophy [1
]. The mechanism of cell death involves, on the one hand, the activation of several apoptotic pathways [22
] and on the other hand the necrotic cell death, probably caused by glutamate-mediated excitotoxicity [2
]. The differential response between mature and immature motoneurons following injury is attributed to the quantity of glutamate receptors on the cell membrane [23
Administration of an NMDA or AMPA/Kainate receptor antagonist within this critical period of development is thought to reverse the neurotoxic effects of axotomy and result in increased survival of motoneurons [2
]. Unfortunately, the protective effects for many of these factors are only transient, lasting 2–3
]. Dizocilpine malate (MK-801), an NMDA antagonist, has been used in animal models in vivo with success, in order to prevent motoneuron death after axotomy. It was badly tolerated by rats, however, due to side effects and high mortality [2
]. Furthermore, magnesium, which is known to act as a voltage-dependent blocker of the N-methyl-D-aspartate (NMDA) channel, by coupling with the specific Mg2+
site within the pore of the ion channel [28
], was found to inhibit the death of ventral horn motoneurons and to restore the alteration in contractile properties provoked by axotomy [1
In the present study we assessed the contractile properties and the movement behaviour in rats of different age groups, following neonatal sciatic nerve crush and administration of DAP5. This is a selective NMDA receptor antagonist that competitively inhibits the ligand (glutamate) binding site of NMDA receptors. DAP5 is generally very fast acting as indicated by in vitro preparations, and can block NMDA receptor action at a reasonably small concentration [30
]. Our hypothesis was that, by delivering an agent with a direct action on the NMDA receptor, we would be able to achieve a more profound effect, than the one observed with the indirect action of magnesium. A drawback in our study is that axotomized hindlimbs were not compared with the ones of the opposite side (right), but with those of control animals, thus rendering our observations more vulnerable to interanimal differences. We chose that design, however, to achieve a correlation with the behavioural tests, which necessarily had to entail a control group of animals. Moreover, body weight did not differ among the experimental groups and consequently the differences in tension recordings may be directly ascribed to the muscle changes.
According to our knowledge, this is the first time that DAP5was administered in vivo. Systematic administration of DAP5 has been discouraged by other researchers, due to poor cerebrospinal fluid (CSF) absorption and probable toxic features [17
]. By initially following titration trials, we did not observe any side effects. In all age groups, no significant difference was found between control animals and those that the agent was administered, in both contractile properties and behavioural tests. These results allowed us to conclude that a safe and effective therapeutic profile is evident for the aforementioned drug, at least for the parameters studied.
Apart from reducing the number of surviving motoneurons, axotomy in the early postnatal period alters the contractile properties of limb muscles, as well [3
]. Our results are in accordance with our previous work [1
], as well as other researchers [2
], showing that axotomy severely impairs tension development by the muscle. The main feature in this study is that DAP5 resulted in the recovery of the contractile properties of both muscles, up to the level of control animals, thus fully eliminating the debilitating effect of axotomy. We assume that the direct action of the agent on the NMDA receptor accounts for the improved results.
Concerning the time evolution of contraction, we reconfirmed that immature (P14) muscles are not yet differentiated into fast- or slow-contracting ones and that fast contracting muscles are more severely affected by axotomy [1
]. In the early developmental stages, contraction in both muscles is rather prolonged, with a high fatigue index. In control animals, EDL attains its normal features by adulthood, in the case of axotomy, however, the muscle becomes slow and fatigue resistant. At this point, there was a differentiation concerning our previous work, as DAP5 succeeded in recuperating in a greater degree the original features of the muscle. In agreement with the majority of researchers, it was of no surprise that soleus did not present any alteration in its time course of contraction, as its contractile properties are not significantly altered throughout early postnatal life. Concerning fatigability, normally soleus is converted progressively into a fatigue resistant muscle. This process is halted, in case of axotomy and is partially reversed by DAP5 administration. We also have to point out that differences were statistically significant in most parameters among the various age groups (within the same experimental group), reflecting the fact that all animals underwent the natural course of contractile properties maturation (force augmentation during development).
In order to evaluate the locomotion, a series of tests was applied, which comprised the rotarod, as well as passing along bridges of different width and footprint analysis. The results were in full correlation with the isometric recordings. Rats with axotomy exhibited overt changes in their locomotor behaviour, compared with controls. Treatment with DAP5 improved movement, although the difference with controls was still discernible. Improvement in locomotor behaviour, following DAP5 administration, however, was not as impressive as the one observed in tension recordings. Axotomy provokes a serious sensorimotor disruption, early in development, and coordination of the limbs during walking does not entirely rely on the reinforcement of individual muscles. Adaptive mechanisms are activated to compensate for the lack of function; the injured rat exerts a notifying effort to use the crushed leg and eventually succeeds in walking, nevertheless, with uncontestable difference with respect to the non-injured one. In addition, it seems that some tests are more specific in delineating subtle differences between the different groups. The rotarod and the footprint analysis turned out to evaluate locomotor behaviour in a more efficient way, than the observation and the grading of the gait.
The lack of side-effects seems rather unexpected in our study, compared to what has been described in the literature [32
]. The NMDA receptor contributes to plasticity, neuronal differentiation and synaptogenesis in the developing nervous system [33
]. NMDA antagonists are notorious for causing a multitude of behavioural sequelae. The disfunction of this receptor is frequently considered to contribute to the pathophysiology of schizophrenia, depression, anxiety and, indeed, these agents are usually implemented in the research of several psychotic states [34
]. Moreover, MK-801, one of the most extensively studied drugs in this category, is known to induce long term behavioural disturbances, when administered in neonatal rats [35
]. One could argue that our locomotor models may not be so sensitive to detect this kind of behavioural defect. Furthermore, despite sharing nominally common mechanisms of action and often presumed biological equivalence, the NMDA antagonists present very diverse effects [36
]. Most antagonists used in animal studies, such as MK-801, act via an uncompetitive antagonism, whereas DAP5 utilizes a competitive mode of action. It might be that, this mode of action, in association with the affinity of the receptor, should provide an effective combination which varies among the different substances and explains the magnitude of primary actions as well as the side effects.