Modulation of the Innate Immune Response by NMDA Receptors Has Neuropathological Consequences

Animals

Adult male C3H/HeN mice (body weight, 25-29 gm; Charles River Canada, St. Constant, Québec, Canada), C3H/HeJ mice (Jax Mice; Jackson Laboratory, Bar Harbor, ME), or Sprague Dawley rats (body weight, ∼250 gm; Charles River Canada) were acclimated to standard laboratory conditions (14/10 hr light/dark cycle; lights on at 6:00 A.M. and off at 8:00 P.M.) with ad libitum access to rodent chow and water. Animal breeding and experiments were conducted according to Canadian Council on Animal Care guidelines, as administered by the Laval University Animal Care Committee.

Experimental protocols

Acute intraparenchymal injections. Mice receiving intrastriatal (IS) injections were anesthetized with an intraperitoneal injection of avertin (2,2,2 tribromoethanol; Sigma-Aldrich, St. Louis, MO) and placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA). The right caudate putamen was reached using a small cannula (33 gauge) at the coordinates 0.0 mm anteroposterior, -2.0 mm lateral, and -3.0 mm dorsoventral according to a mouse brain atlas (Paxinos and Franklin, 2001). The animals received an infusion of either sterile pyrogen-free saline (1 μl), LPS (0.5 μg; from Eschericia coli; serotype O55:B5; Sigma L2880), a mixture of LPS (0.5 μg) and MK-801 maleate (1 μg; Sigma), or MK-801 (1 μg) over 2 min by means of a microinjection pump (model A-99; Razel Scientific Instruments, Stanford, CT). Three or four mice were used per group for each time point, for a total number of 96 mice for this experiment.

At different time points, after intraparenchymal injections (6, 24, and 72 hr and 2 weeks), animals were deeply anesthetized via an intraperitoneal injection of a mixture of ketamine hydrochloride and xylazine and then rapidly perfused transcardially with 0.9% saline, followed by 4% paraformaldehyde in 0.1 m borax buffer, pH 9.5, at 4°C. Brains were removed rapidly from the skulls, postfixed for 2-4 d, and then placed in a solution containing 10% sucrose diluted in 4% paraformaldehyde-borax buffer overnight at 4°C. The frozen brains were mounted on a microtome (Reichert-Jung; Cambridge Instruments Company, Deerfield, IL) and cut into 20 μm coronal sections from the olfactory bulb to the end of the medulla. The slices were collected in cold cryoprotectant solution (0.05 m sodium phosphate buffer, pH 7.3, 30% ethylene glycol, and 20% glycerol) and stored at -20°C.

Long-term intraparenchymal infusions. A chronic indwelling cannula was implanted as described previously (Nadeau and Rivest, 2003). Adult male rats were anesthetized with an intraperitoneal injection of a mixture (1 ml/kg body weight) of ketamine hydrochloride (91 mg/kg) and xylazine (9 mg/kg), and the site of injection was reached stereotaxically (David Kopf Instruments). With the incisor bar placed at 3.3 mm below the intraneural line (horizontal zero), the coordinates from bregma for the guide cannula (22 gauge; C313G; Plastic One, Roanoke, VA) were 0.0 mm anteroposterior, -3.0 mm lateral, and -2.8 mm dorsoventral, according to brain atlas (Paxinos and Watson, 1998). The guide cannula was secured with screws and cranioplastic cement [cranioplastic powder (Plastic One); Dentsply repair material (Dentsply International, York, PA)]. The rats were then housed individually for a 10 d recuperation period. During the first 3 d after the surgery, rats received once daily a subcutaneous injection of 8 ml of Ringer's lactate (Abbott Laboratories, Saint-Laurent, Canada) and 50 μl of ketoprofen (Rhône Mérieux Canada, Victoriaville, Canada). After the recovery period, a mini-osmotic pump (Alzet model 2004; Durect Corporation, Cupertino, CA), connected to an internal cannula (28 gauge; 14 mm long from the pedestal; C313I; Plastic One) with Intramedic polyethylene tubing (PE-50; inner diameter, 0.58 mm; outer diameter, 0.965 mm; Dow Corning, Midland, MI), was implanted subcutaneously in the interscapular region. The internal cannula was connected to the guide cannula, reaching the dorsoventral coordinate at -5.0 mm. The pumps (lot 10047-02; pumping rate, 0.29 μl/hr) were filled with vehicle solution (sterile saline), LPS (0.0718 μg/μl; yielding 0.5 μg/d), a mixture of LPS and MK-801 (LPS, 0.0718 μg/μl; MK-801, 0.1437 μg/μl; yielding 1 μg/d), or MK-801 (0.1437 μg/μl) and incubated at 37°C in sterile saline solution 48 hr before the implantation in vivo. The animals were killed 72 hr after implantation of the mini-osmotic pumps. Three or five rats per group were used for the intraparenchymal infusion of saline (n = 3), LPS (n = 5), LPS plus MK-801 (n = 5), or MK-801 (n = 3). Brain preparation was performed as described above, except for the coronal sections that were cut at 30 μm thickness.

cRNA probes and in situ hybridization

Plasmids were linearized, and the sense and antisense riboprobes were synthesized as described in Table 1. Radioactive cRNA copies were synthesized by incubation of 250 ng of linearized plasmid in 6 mm MgCl₂, 40 mm Tris, pH 7.9, 2 mm spermidine, 10 mm NaCl, 10 mm DTT, 0.2 mm ATP/GTP/CTP, 100 μCi of α -³⁵S-UTP (catalog number NEG 039H; DuPont-NEN, Boston, ME), 20 U of RNAsin (Promega, Madison, WI), and 10 U of T7, SP6, or T3 RNA polymerase for 60 min at 37°C (Table 1). Unincorporated nucleotides were removed using the ammonium acetate precipitation method; 100 μl of DNase solution (1 μl DNase, 5 μl of 5 mg/ml tRNA, and 94 μl of 10 mm Tris/10 mm MgCl₂) was added, and, 10 min later, a phenol-chloroform extraction was performed. The cRNA was precipitated with 80 μl of 5 m ammonium acetate and 500 μl of 100% ethanol for 20 min on dry ice. The pellet was dried and ressuspended in 50 μl of 10 mm Tris/1 mm EDTA. A concentration of 10⁷ cpm probe was mixed into 1 ml of hybridization solution (500 μl formamide, 60 μl of 5 m NaCl, 10 μl of 1 m Tris, pH 8.0, 2 μl of 0.5 m EDTA, pH 8.0, 50 μl of 20× Dehart's solution, 200 μl of 50% dextran sulfate, 50 μl of 10 mg/ml tRNA, 10 μl of 1 m DTT, and 118 μl of DEPC water minus volume of probe used). This solution was mixed and heated for 10 min at 65°C before being spotted on slides.

Hybridization histochemical localization of TLR2, inhibitory factor κBα (IκBα), TNF-α, cluster of differentiation 14 (CD14), inducible nitric oxide synthase (iNOS), IL-12 p40, interferon (IFN)-γ, and NMDAR1 mRNA was performed on every 12th section of the entire rostrocaudal extent of each brain, as described previously (Laflamme et al., 1999). The sections were exposed at 4°C to x-ray films (Biomax; Kodak, Rochester, NY) for 1-3 d. The slides were thereafter defatted in xylene, dipped in NTB-2 nuclear emulsion (diluted 1:1 with distilled water; Kodak), exposed for 15 d (iNOS, IL-12, p40, and IFN-γ transcripts), 13 d (TLR2 and CD14 transcripts), 10 d (TNF-α), 7 d (IκBα transcript), or 4 d (NMDAR1 transcript). The slides were then developed in D19 developer (Kodak) for 3.5 min at 14-16°C, washed for 15 sec in water, and fixed in rapid fixer (Kodak) for 5 min. Tissues were thereafter rinsed in running distilled water for 1 hr, counterstained with thionin (0.25%), dehydrated through graded concentrations of alcohol, cleared in xylene, and coverslipped with distrene plasticizer xylene (DPX) mounting media (Electron Microscopy Science, Washington, PA).

Detection of neuronal cell death, demyelination, and histological analysis

The presence of neuronal cell death was investigated with the Fluoro-Jade B (FJB) method. Briefly, every 12th section of the whole rostrocaudal extent of each brain was mounted onto poly-l-lysine-coated slides, dried under vacuum for 2 hr, dehydrated through graded concentrations of alcohol (50, 70, and 100%; 1 min), rehydrated through graded concentrations of alcohol (100, 70, and 50%; 1 min), and rinsed for 1 min in distilled water. Then, the sections were dipped and shacked in potassium permanganate solution (0.06%) for 10 min and rinsed for 1 min in distilled water. Slides were next dipped and shacked into a solution containing a mixture of 0.0004% FJB (Histochem, Jefferson, AR) plus 0.1% acetic acid (Sigma-Aldrich) plus 0.0002% 4′,6′-diamidino-2-phenylindole (Molecular Probes, Eugene, OR) for 20 min. The slides were thereafter rinsed three times in distilled water (1 min each), dried, dipped in xylene three times (2 min each), and coverslipped with DPX.

Demyelination was determined via Luxol Fast Blue (LFB) staining. The brain sections mounted onto poly-l-lysine-coated slides were dehydrated through graded concentrations of alcohol (50, 70, and 95%; 1 min each) and incubated at 60°C for 6 hr in LFB solution [1% Solvent Blue 38 (Sigma) in 95% ethanol and 0.5% acetic acid]. The sections were then rinsed in 95% ethanol (1 min), 0.05% lithium carbonate (1-5 min; Sigma), and 70% alcohol (two dips). Thereafter, the slides were stained in 1% eosine Y solution (EM Diagnostic System, Gibbstown, NJ) for 30 sec, rinsed in water, incubated in 0.25% cresyl violet (Sigma) for 30 sec, rinsed in water, dehydrated through graded concentrations of alcohol (50, 70, 95, and 100%; 1 min each), cleared in xylene for 1 min (two times), and coverslipped with DPX.

Nissl stain was used as a general index of cellular morphology that may be altered in response to the different treatments.

Quantitative analysis

Quantitative analyses were performed as described previously (Nadeau and Rivest, 2003). Hybridization signals were quantified on x-ray films (Biomax). Briefly, transmittance values (optical density) of the hybridization signal were measured under a Northern Light Desktop Illuminator (Imaging Research) using a Sony Camera Video System attached to a Micro-Nikkor 55 mm-Vivitar extension tube set coupled with a computer and NIH Image software version 1.59/ppc [written by W. Rasband (National Institutes of Health, Bethesda, MD) and available from the internet by anonymous ftp from http://rsb.info.nih.gov/nih-image/download.html]. OD values for each pixel were calculated using a known standard of intensity and distance measurements from a logarithmic specter adapted from Bioimage Visage 110s (Millipore, Ann Arbor, MI). The entire hemisphere ipsilateral to the injection site was digitized and subjected to densitometric analysis, yielding measurements of mean density per area. The OD of each hemisphere was then corrected for the average background signal by subtracting the OD of area without positive signal located in the contralateral side. Four different brain sections at the level of caudate putamen were used for each animal.

The dorsal basal ganglia and hippocampus were selected for the semiquantification of NMDAR1 mRNA. These structures exhibited high constitutive NMDAR1 expression levels and strong transcriptional activation of immune-related genes in response to intracerebral LPS/MK-801 administration. Measurements were performed in 10 C3H/HeN and 10 C3H/HeJ mice, and data are reported as mean OD values (±SEM).

Demyelination was evaluated on digitalized LFB-stained sections within the corpus callosum and caudoputamen as described previously (Nadeau and Rivest, 2003). The rationale to measure LFB staining (index of myelinated fibers) in these regions is based on the pattern of the inflammatory wave that diffused across the dorsal basal ganglia and corpus collosum in response to cerebral infusions. Briefly, OD of corpus callosum and caudato putamen was measured using the NIH Image software, and values of the contralateral side were subtracted from those of the ipsilateral side of four different rostrocaudal levels and expressed as absolute values (mean ± SEM).

The statistical analysis was performed by a one- or two-way ANOVA, followed by a Bonferroni/Dunn test procedure as post hoc comparisons or Student's t test for NMDAR1 subunit mRNA levels.

LPS-induced expression of TLR2 and CD14 is modulated by MK-801 in a TLR4-dependent manner

Figure 1 depicts representative hybridization signals in the brain of mice treated with saline, LPS, LPS/MK-801, or MK-801 alone. As expected, implantation of the cannula and the stress of infusion caused a low expression of TLR2 mRNA. Such a localized pattern of positive TLR2-expressing cells was found in the brain of both saline- and MK-801-infused mice (Fig. 1). This low signal contrasts with the intense and widespread transcriptional activation of TLR2 ipsilateral to the side of LPS infusion (Fig. 1Ab, LPS). MK-801 exacerbated the effects of endotoxin injected directly within the basal ganglia. At 24 hr, the signal was more intense in the brain of mice treated with the NMDAR antagonist and endotoxin (Fig. 1Ac). Semiquantitative analyses performed on x-ray films revealed a significant difference between the LPS- and LPS/MK-801-treated groups (Fig. 1C).

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Figure 1.

TLR2 mRNA expression in the brain of C3H/HeN and C3H/HeJ mice after an intraparenchymal bolus of saline, LPS, or the antagonist of the NMDAR MK-801. A, Representative hybridization signals in emulsion-dipped coronal sections (20 μm) taken from C3H/HeN mice that received only saline solution (a), LPS (b; 0.5 μg), a mixture containing LPS (0.5 μg) and MK-801 (c; 1 μg, LPS/MK-801), or MK-801 (d; 1 μg) in the dorsal basal ganglia. B, Emulsion-dipped coronal sections of C3H/HeJ mice, which bear a loss of function in the TLR4, killed 24 hr after a single bolus of saline solution (a), LPS (b), LPS combined with MK-801 (c), and MK-801 (d). C, Semiquantitative analysis of TLR2 mRNA levels (OD) in the ipsilateral side of C3H/HeN and C3H/HeJ mice 24 hr after the intracerebral insults. Please note the robust hybridization signal across the ipsilateral side of mice that received LPS combined with the antagonist of the NMDARs. Results represent means ± SEM of three to four mice per group. Statistical analysis was performed by using a two-way ANOVA, followed by a Bonferroni's multiple comparison test. ^**Significantly different (p < 0.01) from saline-injected group; ^***significantly different (p < 0.001) from saline-injected group; #significantly different (p < 0.05). For more details, see Materials and Methods. CPu, Caudate putamen; LV, lateral ventricle. Scale bar, 1250 μm.

This amplification by MK-801 was essentially abolished in C3H/HeJ mice, because these mice were much less sensitive to either LPS alone or combined with the NMDAR antagonist (Fig. 1B). It is interesting to note that despite the lack of functional TLR4, a single intracerebral bolus of LPS was able to increase TLR2 expression in regions ipsilateral to the injection site. However, the signal was, to a great extent, lower in C3H/HeJ mice than in their wild-type controls, and MK-801 remained without significant effect in TLR4-mutant mice (Fig. 1B,C). These data indicate that the ability of glutamate and NMDARs to modulate the innate immune response in the brain is dependent on the prior binding of LPS to its receptor TLR4.

The same phenomenon took place for the gene encoding CD14 (Fig. 2). The endotoxin caused a profound increase in the transcriptional activation of the gene encoding LPS receptor in the ipsilateral side of mice killed 6 hr after the single intraparenchymal infusion. The cells lining the leptomeninges and few blood vessels exhibited a strong hybridization signal for CD14 transcript 6 hr after the cerebral injection with LPS alone or combined with MK-801 (Fig. 2b,c). As for TLR2, the expression wave across the brain parenchyma increased in intensity 24 hr after the treatment combining LPS with the antagonist of the NMDAR (Fig. 2k). This effect of MK-801 persisted up to 72 hr after injection, but the differences in the hybridization signal were not as marked as those found at 24 hr (Fig. 2j,k,r s). Once again, C3H/HeJ mice were relatively resistant to the treatments, but the endotoxin was capable of causing an increase in CD14 gene expression in a localized area ipsilateral to the injection site at 6 hr (Fig. 2f,g). The signal largely decreased in the brain of these mice 24 hr after the intracerebral administration of the endotoxin that was combined or not with MK-801 (Fig. 2n,o). Seventy-two hours after the different treatments, very few positive CD14-expressing cells were found in the CNS of CH3/HeJ mice (Fig. 2v,w).

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Figure 2.

Time-related expression of CD14 mRNA in the brain of C3H/HeN and C3H/HeJ mice after a single intraparenchymal bolus of LPS and MK-801. These dark-field photomicrographs were taken from coronal sections hybridized with radioactive CD14 cRNA probe and dipped into nuclear emulsion milk. These images are representative examples of the expression pattern of CD14 transcript 6 hr (a-h), 24 hr (i-p), and 72 hr (q-x) after the infusion with saline, LPS, LPS plus MK-801, or MK-801 alone. Please note the robust signal in the area ipsilateral to the injection site of C3H/HeN mice challenged with LPS and MK-801. Although this effect was impaired in HeJ mice, the endotoxin was still capable of causing a significant increase in CD14 transcription in the brain of this strain (f, g). bv, Blood vessels; cc, corpus callosum; CPu, caudate putamen; Cx, cortex. Scale bar, 1000 μm.

TLR4-dependent induction of proinflammatory signaling and gene transcription

The next series of assays evaluated whether expression of receptors involved in the innate immune response is associated with an increase in NF-κB signaling and transcriptional activation of genes encoding molecules involved in neurodegenerative disorders. The wave of TNF-α-expressing cells was characterized by strong signals at the edges of the inflammatory area restricted to the ipsilateral side 6 hr after LPS or LPS plus MK-801 infusions (Fig. 3b,c). At this time point, C3H/HeJ mice were relatively resistant to the endotoxin, although scattered positive TNF-α cells were detected in the infused region (Fig. 3f,g). Surprisingly, a robust hybridization signal was found in the brain of both mouse strains 24 hr after the single IS bolus of LPS (Fig. 3j,k,n,o). The message vanished quickly in the brain of C3H/HeJ mice (Fig. 3v,w), but remained positive and scattered across the injection site of C3H/HeN animals at 72 hr (Fig. 3r,s). The endotoxin is, therefore, able to trigger transcriptional activation of TNF-α in a very transient manner without a functional TLR4. Here, the effects of the antagonist of the NMDARs were not as convincing as for the other transcripts. However, TNF-α mRNA levels were, in general, higher in the CNS of most C3H/HeN mice treated with LPS and MK-801 and killed 6 hr afterward (Fig. 3c).

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Figure 3.

Expression wave of the proinflammatory cytokine TNF-α in response to intrastriatal infusion of LPS and MK-801. Representative nuclear emulsion dipped coronal sections depicting the effects of LPS and MK-801 on TNFα gene expression in C3H/HeN and C3H/HeJ mouse brains at 6 hr (a-h), 24 hr (i-p), and 72 hr (q-x) after injections. Please note the rapid expression of the gene encoding the cytokine in the brain of C3H/HeN mice after coadministration of LPS and MK-801. The endotoxin also stimulated TNF-α gene expression in the CNS of C3H/HeJ mice, especially at 24 hr (n, o). CPu, Caudate putamen. Scale bar, 1000 μm.

De novo induction of IκBα mRNA is a reliable index of NF-κ B activity, and a strong hybridization was detected in the CNS of C3H/HeN mice (Fig. 4A). Indeed, IS infusion of LPS caused a diffuse pattern of positive cells across the brain parenchyma. The signal was already strong at 6 hr and remained positive up to 72 hr after LPS infusion (data not shown). The combined administration of LPS with the inhibitor of the NMDARs provoked a strong and widespread expression of IκBα mRNA in cells lining the ventricle ependyma, leptomeninges, blood vessels, and across the brain parenchyma ipsilateral to the injection site. The hybridization signal was clearly higher in the cerebral tissues of C3H/HeN mice treated with LPS and MK-801 than those of mice challenged only with the endotoxin and killed 6 hr afterward (Fig. 4Ab,c). It is interesting to note that mice killed 24 hr after the various treatments still exhibited a similar pattern of IκBα-expressing cells, but the message returned to a comparable level at 72 hr (data not shown). Although individual variations were observed among animals of that group, MK-801 was able to increase IκBα expression in response to LPS at 6 and 24 hr. IκBα mRNA levels were barely detectable in the brain of C3H/HeJ mice, which indicates that exacerbation of the proinflammatory signaling by MK-801 depends on a functional TLR4.

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Figure 4.

TLR4-dependent effects of MK-801 on LPS-induced IκBα and iNOS gene expression. A, Dark-field photomicrographs taken from coronal sections hybridized with radioactive IκBα cRNA probe and dipped into nuclear emulsion milk. These images are representative examples of the expression pattern of IκBα transcript (used here as an index of NF-κB activity) (a-h) 6 hr after the infusion with saline, LPS, LPS plus MK-801, or MK-801 alone. B, Positive signals for iNOS mRNA were only detected in C3H/HeN mice treated with LPS or LPS plus MK-801 (j and k, respectively) 6 hr after the infusion. The cerebral tissues of C3H/HeJ mice failed to show any positive signal for the mRNA encoding iNOS. bv, Blood vessels; cc, corpus callosum; chp, choroid plexus; CPu, caudate putamen; LV, lateral ventricle. Scale bar, 1000 μm.

Unlike the other transcripts, no positive signal was found for the gene encoding iNOS in the brain of C3H/HeJ mice in response to the endotoxin injected either alone or together with MK-801 (Fig. 4B). In C3H/HeN mice, the message significantly increased over the background level 6 hr after the intracerebral infusion of LPS alone or combined with MK-801 (Fig. 4Bj,k). At 24 hr, numerous clusters of iNOS-expressing cells were detected in the region infused with endotoxin, and the antagonist of the NMDARs failed to modulate the pattern of iNOS expression that was nonuniform and restricted to isolated clusters of positive cells (data not shown). The signal returned to background levels 72 hr after the IS infusion with the bacterial cell wall component combined or not with MK-801 (data not shown).

NMDAR1 subunit expression in the brain of C3H/HeN and C3H/HeJ mice

To verify whether the NMDA antagonist failed to modulate LPS-induced immune response in C3H/HeJ mice because of a major difference in receptor expression, NMDAR1 subunit mRNA was measured via in situ hybridization histochemistry. As shown in Figure 5, the pattern and intensity of NMDAR1 gene expression were similar in the brain of both mouse strains. The distribution of the gene encoding this critical NMDAR was also similar to that already described in other studies (Laurie and Seeburg, 1994). Semiquantitative analyses were performed in caudate putamen and hippocampus because of the high expression levels and well-defined pattern of the receptor transcript in these structures. C3H/HeN and C3H/HeJ mice exhibited similar NMDAR1 mRNA levels in different regions of the brain (Fig. 5C).

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Figure 5.

Expression of NMDAR1 subunit mRNA in the brain of C3H/HeN and C3H/HeJ mice. A, Representative signals in coronal sections hybridized with sense or antisense riboprobe and exposed to x-ray films (Biomax; Kodak). B, Dark-field photomicrographs taken from coronal sections hybridized with radioactive NMDAR1 cRNA probe and dipped into nuclear emulsion milk. Intensity and pattern of the hybridization signal were similar across the brains of C3H/HeN and C3H/HeJ mice. C, Semiquantitative analysis of relative NMDAR1 mRNA levels (OD) in the caudate putamen (CPu) and hippocampus (Hip) of C3H/HeN and C3H/HeJ mice. Scale bar, 1000 μm.

Integrity of the neuronal elements

The consequences of altering the inflammatory response by MK-801 and TLR4 on the cerebral tissue were determined via two different approaches. FJB is a polyanionic fluorescein derivative that has been shown to be a sensitive and reliable marker for histochemical localization of neuronal degeneration (Schmued et al., 1997). Brain sections of animals with bilateral adrenalectomy were used as positive control, because a subpopulation of cells within the dentate gyrus granular layer are known to undergo neuronal cell death under this treatment. These neurons displayed strong and selective fluorescent FJB signals in the sections of a rat killed 6 d after being adrenalectomized (Fig. 6Aa). Such positive cells were never observed in the brain of LPS- or LPS plus MK-801-treated C3H/HeN mice 24 or 72 hr after infusions (Fig. 6Ab-g), except for the neurons adjacent to the cannula track. FJB neurons along the tract were found in all animals, including those infused only with vehicle solutions (Fig. 6Ab). Few capillaries of LPS-treated mice contained small and scattered positive cells, which are likely to be infiltrating cells (Fig. 6Ad-f, insets). Neurons remained negative for the fluorochrome FJB up to 2 weeks after the different treatments in both C3H/HeN and C3H/HeJ mice.

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Figure 6.

Neuronal integrity in the brain of C3H/HeN and C3H/HeJ mice after intrastriatal infusion of LPS and the antagonist of NMDARs. A, The fluorochrome FJB was used as a marker for histochemical localization of degenerative neurons. Degenerating neurons (positive control) were found in hippocampus of a rat killed 6 d after being adrenalectomized (a, inset). Such positive neurons were never observed in regions depicting the robust expression of the different transcripts, except along the needle track because of the mechanical injury (white arrowheads). Small non-neuronal cells (probably infiltrating cells) were also found in the CNS of C3H/HeN mice treated with LPS alone or combined with MK-801 (white arrows). B, Nissl-stained sections at the level of dorsal basal ganglia. Neuronal density and morphology were similar among all the groups included in this study. Black arrows point to the cannula track. Cx, Cortex; cc, corpus callosum; contra, contralateral side; CPu, caudate putamen; ipsi, ipsilateral side; ct, cannula track. Scale bars, 100 μm; insets, 50 μm.

Nissl stain was also used to evaluate the morphology and histological integrity of the cerebral tissues. Figure 6B shows examples of brain sections taken from saline-, LPS-, or LPS plus MK-801-infused mice, and no convincing changes were observed 72 hr after the different treatments (Fig. 6Ba-c). The sections remained intact, and ipsilateral sides were similar to the contralateral areas at 2 weeks after LPS infusion in both mouse strains (Fig. 6Bd-f).

Genes involved in the transfer from the innate to adaptive immunity

The gene encoding TLR2 was still expressed in the brains of C3H/HeN mice 2 weeks after the single bolus of LPS (Fig. 7a) but not in those of C3H/HeJ mice (Fig. 7b). Few positive and small scattered IκBα- and TNF-α-expressing cells were also found, but the cerebral tissues of all groups remained without detectable signal for both IL-12 and IFN-γ transcripts. Positive controls for these two genes were hybridized in the brains of mice infected with herpes simplex virus type 2, which causes a profound neurodegeneration associated with innate and adaptive immune responses (Boivin et al., 2002).

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Figure 7.

The innate immune response is not associated with a transfer to the adaptive immunity. Representative examples of nuclear emulsion-dipped coronal sections depicting expression of genes involved in the transfer from the innate to adaptive immune response 2 weeks after LPS injection in the dorsal striatum. TLR2 mRNA was still expressed in the ipsilateral side of C3H/HeN mice 14 d after a single bolus of LPS (a), whereas such signal was not present in the infused regions of C3H/HeJ mice (b). Few IκBα- and TNF-α-expressing cells were also found in the brain of C3H/HeN mice (c, d, white arrows). In situ hybridization failed to detect any positive signal for IL-12 and IFN-γ across the cerebral tissue of C3H/HeN mice (e and f, respectively). cc, Corpus callosum; CPu, caudate putamen. Scale bar, 1000 μm.

Brain damage caused by a long-term infusion

To verify the relevance of innate immune response modulated by glutamate to brain physiopathology, an experimental protocol was designed to expose the cerebral tissue to long-term stimulation. Figure 8 depicts LFB-stained sections from animals treated with saline, LPS, LPS combined with MK-801, or MK-801 alone during 72 hr. Animals that received combined treatment of LPS plus MK-801 exhibited a striking degree of demyelination compared with the other groups, which was confirmed by semiquantitative analysis of differential OD with the contralateral side (Fig. 8A,B). LPS infusion for a period of 3 d was also able to cause demyelination, but differential OD failed to reach statistical significance because of the high degree of variability among animals of this group (Fig. 8A,B). It is possible that variable diffusions throughout the brain parenchyma contribute to different degrees of demyelination between animals and dorsoventral-rostrocaudal levels. However, demyelination was clear in all animals that received the double treatment, which was not the case in the cerebral tissue of rats that were infused only with the endotoxin. Demyelination was apparently not associated with neuronal cell death, because no positive FJB neurons were found in the brain of chronically treated rats. This pattern of demyelination was never observed in the brain of C3H/HeN or C3H/HeJ mice that received an acute bolus with the different solutions (data not shown).

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Figure 8.

Demyelination in response to long-term infusion of the endotoxin LPS and NMDAR antagonist MK-801 in ipsilateral sides of adult male SD rats. A, Luxol Fast Blue-stained coronal sections from animals infused with saline, LPS (0.5 μg/d), LPS combined with MK-801 (1 μg/d), or MK-801 alone and killed 72 hr after implantation of mini-osmotic pumps. B, Differential OD measured in several rostrocaudal sections of each rat included in this experiment. Statistical analysis was performed by using a one-way ANOVA, followed by a Bonferroni's multiple comparison test. ^*Significantly different (p < 0.01) from the saline-injected group. For more details on image analysis, see Materials and Methods. Black arrowheads indicate the tip of the cannula. Scale bar, 1250 μm.