Upon PV infection, hPVR-tg mice develop a paralytic condition clinically and pathologically reminiscent of primate poliomyelitis (9
). Four experimental groups of hPVR-tg mice were used: group I, untreated hPVR-tg mice; group II, mice that were treated with a left sciatic nerve transection alone (Fig. ); group III, sham-operated mice that received multiple i.m. injections at intervals indicated in the legend to Fig. ; and group IV, mice subjected to a left-sciatic-nerve transection that were treated with the same regimen of multiple i.m. injections as group III (Fig. ).
FIG. 1 Implementation of the neural block. The sciatic nerve was severed, disconnecting its peripheral branches, the common peroneal and tibial nerves, from the spinal cord. Motoric and sensory innervation of the gastrocnemius muscle in the limb treated was (more ...)
FIG. 2 Clinical course of PPM in hPVR-tg mice infected with PV1(M). hPVR-tg mice of four experimental groups were infected with 5 × 106 PFU of PV1(M) by the intravenous route. At defined intervals following virus inoculation (2 h, 6 h, 24 h, and 48 h (more ...)
Sciatic nerve transections were performed in such a manner as to minimize muscle trauma interfering with the experiment (Fig. and Materials and Methods). Sham-operated mice were subjected to the identical procedure, only the nerve transection itself was left out. Seven days after surgery, all mice were inoculated intravenously with 5 × 106 PFU of PV1(M). Mice from groups III and IV were subsequently given i.m. injections into the left gastrocnemius muscle at regular intervals (Fig. ). At 48, 72, and 96 h postinfection (p.i.), four animals from each experimental group were sacrificed, and the following analyses were carried out. (i) At the time of sacrifice, a clinical neurological status was established. (ii) Tissues from the cerebral cortex, cervical spinal cord, lumbosacral spinal cord, injected gastrocnemius muscle (where applicable), uninjected skeletal muscle, and serum were obtained. All tissues were homogenized and the virus load in the homogenate was quantified in a plaque assay. (iii) Tissue specimens from four areas of the spinal cord were obtained and analyzed histopathologically.
Clinical evaluation revealed an increased susceptibility to PV-induced neurological complications due to muscle injury inflicted by multiple i.m. injections (Fig. ). Group III mice developed more severe signs of poliomyelitis earlier than their peers from group I (Fig. ). The onset of paralysis in animals of group III occurred preferentially in the injected lower extremity, whereas animals of groups I and II as well as from group IV generally developed paraparesis initially (Table ). Interruption of the peripheral nerve connection between the site of i.m. injections (gastrocnemius muscle) and the ipsilateral spinal cord protected mice from aggravation of the clinical course of poliomyelitis induced by multiple i.m. injections (group IV [Fig. ]). This was evidenced by the slow course of clinical progression in infected animals that had been treated with a sciatic nerve transection prior to the administration of i.m. injections. The nerve transection itself did not influence the course of progression of poliomyelitis, since untreated control mice showed progression of neurological deterioration at the same rate as mice from group II (Fig. ).
TABLE 1 Sites of initial paralysis in PV-infected hPVR-tgmicea
Analyses of tissues of hPVR-tg mice revealed two factors involved in the provocation-induced, aggravated clinical course of poliomyelitis. Firstly, whereas skeletal muscle scarcely produces PV progeny in viremic animals (8
), virus growth was stimulated in muscle tissue that had been injured by repeated i.m. injections (Fig. A). Secondly, in sham-operated mice treated with multiple i.m. injections, virus replication in lower segments of the spinal cord was significantly accelerated and elevated over propagation levels in the same area in nerve-transected animals (Fig. B). In contrast, replication rates in different experimental groups were less divergent among the same cervical segments of the spinal cord (Fig. C). No viral replication beyond the load of the original inoculum was detected in homogenates of tissues from the cerebral cortex and in serum.
FIG. 3 Replication profiles of PV1(M) in tissues of hPVR-tg mice. Mice were sacrificed at the indicated time points following intravenous virus administration. (A) Replication rates in gastrocnemius muscle. Virus replication in uninjured skeletal muscle from (more ...)
Enhanced virus replication within traumatized muscle occurred independently of sciatic nerve transection as evidenced by its occurrence to the same extent in animals derived from groups III and IV (Fig. A). This differs from the nature of viral propagation within the lower spinal cord in animals with sciatic nerve transection and mice with intact sciatic nerves (Fig. B). In the former, replication rates in the lumbosacral spinal cord were much lower than in the same region of the spinal cords of mice that were sham operated only.
From these observations it is apparent that the provocation effect of muscle injury is based on the induction of PV entry into the peripheral nerve and retrograde axonal transport to the CNS. Facilitated access to the CNS by retrograde axonal transport induced accidentally by muscle injury may shorten the incubation period, localize the initial paralytic symptoms, and hasten the progress of virus replication during PPM. This is strongly supported by the observation that peripheral nerve transection interrupting the neural connection between injured muscle and the spinal cord protects mice from PPM (Fig. ). We observed an increase in viral replication within injured skeletal muscle; in this muscle only small quantities of virus were produced, but the increase may have enhanced the efficiency of PV uptake into the peripheral nerve. Although muscle injury in animals from group IV caused an increase in viral replication, the enhancement of proliferation did not extend to the spinal cord (Fig. B).
The conclusions based on clinical observations and quantitation of viral replication in specific tissues were confirmed by histopathological analysis. Spinal cord tissue specimens were harvested from sacrificed mice at given time points and processed for histopathological examination (Fig. ). The sequence of images demonstrates the progression of poliomyelitic lesions within the lumbosacral spinal anterior horn in hPVR-tg mice. Mice from experimental groups I and II developed lesions typically seen in poliomyelitis only after 96 h p.i. (Fig. , panel 1C). Treatment of mice with multiple i.m. injections accelerated the progression of spinal cord lesions induced by PV. In mice from experimental group III, complete destruction of the motor neuron population was already achieved by 72 h p.i. (Fig. , panel 2B). At this time, no histopathological damage was evident in the control group (Fig. , panel 1B). The increase in the rate of appearance of histological lesions due to repeated i.m. injections covaried with the aggravated clinical course as well as the elevation of virus titers in tissues targeted by PV in mice of group III. As is evident, sciatic nerve transection protected hPVR-tg mice from the aggravated progression of poliomyelitic lesions. In mice from group IV, the pace of advance of poliomyelitic pathology was synchronous with the rate of progression in the control group (Fig. ; compare panels 1A to C with panels 3A to C). Sections obtained from thoracocervical spinal segments showed a less evident advance of lesion progression in mice from group III (data not shown). This corresponded to the minor increase in viral replication in cervical regions of the spinal cords of mice from group III (Fig. C).
FIG. 4 Histopathological evaluation of PV-induced damage within the lumbosacral segments of the spinal cords of mice from experimental groups I (panels 1A to C), III (panels 2A to C), and IV (panels 3A to C). The results of histopathological analysis of tissue (more ...)