Pre-existing Toxoplasma gondii infection increases susceptibility to pentylenetetrazol-induced seizures independent of traumatic brain injury in mice

Abstract

Introduction: Post-traumatic epilepsy (PTE) is a debilitating chronic outcome of traumatic brain injury (TBI), and neuroinflammation is implicated in increased seizure susceptibility and epileptogenesis. However, how common clinical factors, such as infection, may modify neuroinflammation and PTE development has been understudied. The neurotropic parasite, Toxoplasma gondii (T. gondii) incurably infects one-third of the world's population. Thus, many TBI patients have a pre-existing T. gondii infection at the time of injury. T. gondii infection results in chronic low-grade inflammation and altered signaling pathways within the brain, and preliminary clinical evidence suggest that it may be a risk factor for epilepsy. Despite this, no studies have considered how a pre-existing T. gondii infection may alter the development of PTE.

Methods: This study aimed to provide insight into this knowledge gap by assessing how a pre-existing T. gondii infection alters susceptibility to, and severity of, pentylenetetrazol (PTZ)-induced seizures (i.e., a surrogate marker of epileptogenesis/PTE) at a chronic stage of TBI recovery. We hypothesized that T. gondii will increase the likelihood and severity of seizures following PTZ administration, and that this would occur in the presence of intensified neuroinflammation. To test this, 6-week old male and female C57BL/6 Jax mice were intraperitoneally injected with 50,000 T. gondii tachyzoites or with the PBS vehicle only. At 12-weeks old, mice either received a severe TBI via controlled cortical impact or sham injury. At 18-weeks post-injury, mice were administered 40 mg/kg PTZ and video-recorded for evaluation of seizure susceptibility. Fresh cortical tissue was then collected for gene expression analyses.

Results: Although no synergistic effects were evident between infection and TBI, chronic T. gondii infection alone had robust effects on the PTZ-seizure response and gene expression of markers related to inflammatory, oxidative stress, and glutamatergic pathways. In addition to this, females were more susceptible to PTZ-induced seizures than males. While TBI did not impact PTZ responses, injury effects were evident at the molecular level.

Discussion: Our data suggests that a pre-existing T. gondii infection is an important modifier of seizure susceptibility independent of brain injury, and considerable attention should be directed toward delineating the mechanisms underlying this pro-epileptogenic factor.

Keywords: epileptogenesis; immune response; neuroinflammation; oxidative stress; post-traumatic epilepsy (PTE).

FIGURE 1

Study timeline. (A) All mice were injected with either Toxoplasma gondii or vehicle only. Six-weeks were allowed to pass to establish a chronic infection. At this point, mice received either a controlled cortical impact (CCI) or sham injury. After an 18-week recovery (i.e., at 24-weeks from the initial T. gondii or vehicle injection), mice underwent pentylenetetrazol (PTZ) administration for assessment of seizure susceptibility immediately prior to euthanasia and tissue collection. (B) Body weight significantly differed between male and female mice throughout the study, with males weighing more than females. By 6-weeks post-injection, T. gondii mice weighed significantly less than vehicle groups, and this difference was sustained until PTZ administration/endpoint. ^#T. gondii-infected mice significantly differ from vehicle mice, ^@female mice significantly differ from male mice, p < 0.05. n = 10–12/group/sex. Data shown is mean ± SEM.

FIGURE 2

Controlled cortical impact (CCI) results in long-term structural brain damage. Template structural MRI images show chronic brain damage in CCI groups compared to sham groups.

FIGURE 3

Toxoplasma gondii infection increased chronic susceptibility to pentylenetetrazol (PTZ)-induced seizures irrespective of injury. (A) T. gondii and female mice reached higher seizure severity scores compared to vehicle and male groups, respectively. (B) Overall, a higher percentage of mice in T. gondii groups developed generalized convulsive seizures compared to vehicle groups, yet there was no difference between injured and sham groups. (C) Of the mice that exhibited a generalized convulsive seizure, female and T. gondii groups had decreased seizure latency. (D) In addition, T. gondii-infected mice had increased seizure duration compared to vehicle mice. ^#T. gondii-infected mice significantly differ from vehicle mice, ^@female mice significantly differ from male mice, p < 0.05. n = 10–12/group/sex. Data shown is mean ± SEM.

FIGURE 4

Toxoplasma gondii infection increased leukocyte and glial cell gene expression in the ipsilateral cortex of mice chronically post-controlled cortical impact (CCI). Gene expression of GFAP (A), CD45 (B), CD86 (C), CD206 (D), IBA1 (E), TMEM119 (F), TREM2 (G), and CCL2 (H) was increased in T. gondii infected mice compared to vehicle mice. Gene expression of GFAP (A) and CD206 (D) was also increased in mice that received a CCI compared to sham-injured mice. ^#T. gondii-infected mice significantly differ from vehicle mice, *CCI mice significantly differ from sham-injured mice, p < 0.05. n = 4–5/group/sex. Data shown is mean ± SEM.

FIGURE 5

Toxoplasma gondii infection increases gene expression of CD4⁺ and CD8⁺ T-cell markers within the ipsilateral cortex chronically post-controlled cortical impact (CCI). T. gondii-infected mice had increased CXCR3 (A), GATA3 (B), SOCS1 (C), STAT1 (D), CD62L/SELL (E), KLRG1 (F), PRDM1 (G), and FOXP3 (H). On top of this, T. gondii-infected females had increased expression of FOXP3 compared to both T. gondii-infected males and vehicle females. ^#T. gondii-infected mice significantly differ from vehicle mice, ^##T. gondii-infected females significantly differ from T. gondii-infected males, ^###T. gondii-infected females significantly differ from vehicle females, p < 0.05. n = 4–5/group/sex. Data shown is mean ± SEM.

FIGURE 6

Chronic Toxoplasma gondii infection increases gene expression of neuroinflammatory mediators in both sexes. Increased expression of TSPO (A), NLRP3 (B), IL1β (C), TNFα (D), CSF1/M-CSF (E), CSF2/GM-CSF (F), and IFNγ (G) was increased in T. gondii-infected mice compared to vehicle mice. Further to this, T. gondii + controlled cortical impact (CCI) females also had increased expression of CSF2/GM-CSF compared to T. gondii + CCI males, T. gondii + sham females, and vehicle + CCI females. T. gondii + sham males also had increased expression of CSF2/GM-CSF compared to vehicle + sham males. ^#T. gondii-infected mice significantly differ from vehicle mice, ^$T. gondii + sham males significantly differ from vehicle + sham males, ^&T. gondii + CCI males significantly differ from T. gondii + CCI females, % T. gondii + sham females significantly differ from T. gondii + CCI females, *vehicle + CCI females significantly differ from T. gondii + CCI females, p < 0.05. n = 4–5/group/sex. Data shown is mean ± SEM.

FIGURE 7

Chronic Toxoplasma gondii infection increases gene expression of oxidative stress mediators. Gene expression of IDO1 (A), CYBB (B), and NOS2 (C) was increased in T. gondii-infected mice compared to vehicle mice. A sex by infection interaction was detected for the expression of APAF1 (D), however, post-hoc analyses failed to reach significance. ^#T. gondii-infected mice significantly differ from vehicle mice, p < 0.05. n = 4–5/group/sex. Data shown is mean ± SEM.

FIGURE 8

Toxoplasma gondii, controlled cortical impact (CCI), and sex alter the expression of glutamate pathway components. Expression of GLT1 (A) and GAD1 (B) were decreased in T. gondii-infected and female mice compared to vehicle and male mice, respectively. CCI females had lower expression of GLT1 and GAD1 compared to CCI males. In contrast, T. gondii-infected and CCI mice had increased expression of GLAST (C) compared to vehicle and sham mice, respectively. Expression levels of PHGDH (D) were lower in female mice compared to males. ^#T. gondii-infected mice significantly differ from vehicle mice, *sham mice significantly differ from CCI mice, ^@male mice significantly differ from female mice, ^**male CCI mice significantly differ from female CCI mice, p < 0.05. n = 4–5/group/sex. Data shown is mean ± SEM.