To verify that NSV would behave in spinal cord organotypic slice cultures as it does in susceptible mice (Darman et al., 2004
; Jackson et al., 1987
; Nargi-Aizenman et al., 2004
), we have characterized NSV infection of these cultures. After 12 h of incubation in serum-free media, each well (containing 5×250 µm thick slices) of spinal cord organotypic cultures was inoculated with 1×107
PFU/mL administered directly to the tissue slices. Culture supernatants were collected from at least 3 individual wells at each time point. Over a 48-hour period virus levels increase in culture supernatants by several log units () confirming that there is viral replication within this culture system. Since NSV is a neuronotropic virus in vivo
, we investigated whether exclusively neurons were infected in vitro
as well. We found that though astrocytes and microglial cells were not productively infected by a recombinant NSV-GFP construct (), motor neurons () were infected, leading to the expression of GFP within these cells. Additionally, NSV infection of organotypic sections led to cellular injury as defined by LDH release () and loss of motor neurons (). These findings suggested that this culture system replicated the in vivo
tropism and neuronal injury seen with NSV infection in vivo
and that because of the absence of the acquired immune system would allow us to determine intrinsic neural determinants of neural injury following viral infection.
Fig. 1 ñ NSV infection of spinal organotypic cultures induces astrocytic changes and motor neuron death. Spinal cord organotypic cultures were generated from 8-day old Sprague-Dawley rats and infected 1 week after culturing. (A) Viral titers in culture (more ...)
We investigated the humoral inflammatory response within organotypic cultures following NSV infection from the supernatants of at least 3 wells of slice cultures using multiple cytokine/chemokine protein arrays. We found TNF-α and IL-6 consistently elevated at early time points post infection (data not shown). We have previously cytokine/chemokine arrays to screen for inflammatory factors upregulated in NSV-infected spinal cord lysates and found that there was no elevation of multiple factors including IL-2, IL-4, IFN-γ, RANTES and IL-17. We also performed quantitative ELISA for nitric oxide metabolites and MCP-1 and did not see elevation (data not shown).
We then quantitatively defined the time course and extent of this upregulation after NSV infection (). While TNF-α could not be detected at time 0 of infection, it became significantly elevated at 12 and 24 h post infection. Peak TNF-α levels were approximately 900±0.202 pg/mL in the supernatant of infected cultures at 24 h post infection. These studies confirm that an endogenous inflammatory response is initiated within infected organotypic cultures characterized by a cytokine response similar to what has been observed in vivo
(Binder and Griffin, 2001
; Wesselingh et al., 1994
). TNF-α became significantly elevated through time at 12 and 24 h following NSV infection (p
Fig. 2 ñ NSV infection of spinal organotypic cultures elicits and innate inflammatory response. Culture supernatants were sampled at the indicated time points. Supernatants were clarified to eliminate cellular debris and frozen down until used for ELISA. (more ...)
We previously showed that downregulation of the astrocytic glutamate transporter GLT-1 was an important determinant in the outcome from NSV infection in vivo
(Darman et al., 2004
) and so we chose to investigate whether NSV infection alters astrocyte glutamate transport in vitro
. We generated at least 3 pooled tissue lysates from NSV-infected spinal cord organotypic culture slices and carried out immunoblot analyses against GLT-1. As observed in lysates from NSV-infected adult mice (Darman et al., 2004
), there was a significant decrease in GLT-1 protein expression through time over a 24-hour period of NSV-infection of spinal cord organotypic cultures (; p
<0.05). In order to determine if this decline in GLT-1 protein correlated with a decline in functional GLT-1-mediated glutamate transport, membrane preparations were generated from pooled tissues of organotypic slice cultures in triplicate (). Membrane preparations from at least 3 sample pools were exposed to radioactive glutamate in an assay that primarily tests the ability of GLT-1 to mediate glutamate transport (Darman et al., 2004
; Sepkuty et al., 2002
). GLT-1-mediated glutamate transport was significantly reduced in cultures 24 h post-NSV infection (61.727 pmol/mg/min±11.8), compared to mock-treated controls (139.70 pmol/mg/min±15.7; p
<0.05). We conclude that NSV infection of spinal organotypic cultures results in neuronal specific infection, neuron death, and repression of GLT-1 expression and function.
Fig. 3 ñ NSV infection of spinal organotypic cultures induces the downregulation of astrocyte mediated glutamate transport. Samples were collected from NSV-infected cultures at the indicated time points. (A, B) GLT-1 protein expression, normalized to (more ...)
Since TNF-α has been shown to directly downregulate the expression(Fine et al., 1996
; Su et al., 2003
; Szymochaet al., 2000
) and function(Fine et al., 1996
; Zou and Crews, 2005
) of GLT-1,we determined whether purified, recombinant rat TNF-α was capable of mimicking the observed downregulation of GLT-1 seen after NSV infection in spinal organotypic cultures. TNF-α treatment of spinal cord organotypic slice cultures results in a transient increase in GLT-1 protein expression, followed by a repression at 24 h post-treatment (, p
<0.05). GLT-1-mediated glutamate uptake was also significantly reduced (p
<0.05) upon treatment with TNF-α at 24 h post treatment (46.89 pmol/mg/min±5.80) compared to mock treatment (139.7 pmol/mg/min±15.7, ). These data suggest that TNF-α is sufficient to induce a reduction in GLT-1 expression in spinal cord organotypics and may be a critical inflammatory mediator of excitotoxic neuron death following NSV infection.
Fig. 4 ñ TNF-α treatment of spinal organotypic cultures is sufficient to induce downregulation of astrocyte mediated glutamate transport. (A, B) GLT-1 protein expression in TNF-α-treated spinal organotypic cultures at the indicated time (more ...)
In order to determine if TNF-α is necessary for the reduction in GLT-1 expression following NSV infection, we used TNF-α−/− spinal organotypic cultures infected with NSV. Cultures generated from TNF-α deficient mice were susceptible to NSV-infection and replicate virus similarly to cultures generated from wild type mice (). Expression of GLT-1 was preserved in pooled spinal cord organotypic slice cultures deficient in TNF-α (). Resolved bands represent comparisons of tissue collected from individual TNF-α−/− animals that were either mock treated (0) or infected (24, ). Relative ratios of GLT-1 expression in TNF-α−/− (0.9088±0.031) compared to WT (0.6071±0.061, p<0.05) 24 h after NSV infection () reveal preservation of GLT-1 expression in the absence of TNF-α. We found that functional glutamate transport from mouse organotypic sections was unreliable and therefore presented these data in the form of persistence of GLT-1 by immunoblot analyses.
Fig. 5 ñ TNF-α is necessary for NSV-induced downregulation of astrocyte mediated glutamate transport. Spinal organotypic slice cultures were generated from 8-day old TNF-α-deficient mice and infected 1 week after culturing. (A) Viral (more ...)
To test the importance of TNF-α in NSV pathogenesis in vivo
, 5 6 week old TNF-α deficient mice were infected with NSV and the animals were monitored daily for mortality. TNF-α−/− mice were significantly protected from NSV-mediated mortality (mortality of 33% vs 91% for TNF-α−/− vs WT at 7 days post infection, p
<0.05, ). All of the WT control mice were dead by 9 days post infection which is typical for NSV infection (Thach et al., 2000
), however, after 14 days, only 30% of TNF-α KO mice had succumbed to NSV-mediated mortality. Peak virus replication in these animals is similar, though virus persists in the TNF-α deficient mice compared to surviving WT mice (). This reduction in mortality was associated with a paradoxical upregulation of GLT-1 expression at days 3–5 post infection as defined by immunoblot analyses with a return to baseline levels by 8 days post infection (). Consistent with these findings there was a near complete preservation of GLT-1 mediated glutamate transport at 6 days post infection (). These data suggest that TNF-α is necessary to induce the downregulation of GLT-1 expression and function in vivo
following NSV. While we are not sure why there was a paradoxical upregulation of GLT-1 expression at days 3–5 post infection, we have previously reported similar findings when microglial activation was inhibited during NSV infection (Darman et al., 2004
Fig. 6 ñ TNF-α deficient mice are resistant to NSV-induced glutamate downregulation and are protected from NSV-induced death. (A) Survival curves of both TNF-α−/− (n=13) and WT C57BL/6 (n=15) mice demonstrate a protection (more ...)
In order to investigate the disparity in mortality observed between the wild type and TNF-α-deficient mice infected with NSV, 5 animals were given intravenous injections of hydro-ethium (HEt) prior to sacrifice. In dying cells, HEt is taken up and oxidized to a red fluorescent dye, ethidium (Murakami et al., 1998
). Tissues collected from HEt injected mice were cryosectioned and counter stained for neurons with Nissl green. Because the difference in mortality may be related to differential survival of motor neuron populations in the brain stem and cervical spinal cord, we looked for HEt incorporation in these regions and in the hippocampus of NSV-infected WT and TNF-α−/− mice (). There were more HEt-positive cells in the cortex, hippocampus, brain stem and cervical spinal cord of wild type mice compared to TNF-α deficient mice at 5 days post infection ().
Fig. 7 ñ Increased in neuronal death in WT compared to TNF-α deficient mice. Animals were treated with hydroethidium (HEt) prior to sacrifice in order to detect the presence of dying cells. There is more neuronal HEt incorporation in the cervical (more ...)
To determine whether brainstem and cervical motor neurons were resistant to NSV-induced death in the TNF-α−/− mice, we examined and quantified surviving motor neurons in a series of brainstem nuclei and the rostral cervical spinal cord. Neurons in these regions are important in cardiovascular and respiratory control. We reasoned that resistance of these neurons to NSV-induced excitotoxic death might correlate with the enhanced survival of these animals in response to NSV. The ventral respiratory group (VRG) nucleus, hypoglossal (XII) nucleus and the nucleus tractus solitarius (NTS) were chosen both because they are glutamatergically innervated (Schlenker et al., 2001
) and regulate cardiovascular and pulmonary function (Bradley et al., 1996
; Lawrence and Jarrott, 1996
; Smith et al., 1991
; Withington-Wray et al., 1988
). The VRG and XII include neurons responsible for respiration-related motor output and the VRG also includes the pre-Botzinger complex, a likely site for automatic rhythmic breathing (Smith et al., 1991
; Withington-Wray et al., 1988
). The area postrema, a primarily noradrenergic non-glutamatergic system, was used as a control nucleus which would not be expected to be differentially resistant to NSV in WT and NSV animals since it does not receive glutamate as a neurotransmitter.
We found that at baseline (day 0) the number of neuronal nuclei in the area postrema, VRG, XII, NTS and cervical spinal cord did not differ between WT and TNF-α−/− mice. At day 7, there was a significantly reduced number of surviving neurons in the VRG, XII NTS and cervical spinal cord, but not in the non-glutamatergic neurons of the area postrema (). Remarkably, in the TNF-α−/− mice, there was no decline in the number of surviving neurons in any of the four examined brainstem nuclei or the cervical spinal cord. This confirms that there is a significant correlation between animal mortality and death of motor neurons involved in respiratory and cardiovascular control in WT mice. Further, this confirms that TNF-α is required for the death of motor neurons that receive glutamatergic input and that protection of these neurons by the absence of TNF-α correlates with enhanced survival of these mice in response to NSV infection.
ñ Mean number of neurons in each of the brainstem nuclei (the area postrema, the ventral respiratory group, cranial nerve XII and the nucleus tractus solitarius) and motor neurons in the cervical spinal cord
We conclude, therefore, that both in vitro and in vivo, infection with NSV results in upregulation of TNF-α that results in downregulation of GLT-1 expression and function. In the absence of TNF-α, GLT-1 expression is preserved and critical neuronal populations do not die resulting in markedly reduced mortality. We further conclude that excess TNF-α, produced from CNS cells, is critical in NSV pathogenesis and potentiates motor neuron loss.