Appearance, behavior, and neurological findings in the initial cohort
Decrements in behavioral and neurological function in infected mice were first observed in a pilot study and then systematically characterized for all assessments in the "initial cohort" (Table ; Figures , ; movie – Additional file 1
). These results were observed in carefully monitored and characterized SPF mice chronically infected with T. gondii
. Standard measures of appearance (grooming, body position, piloerection, tail wounding, gait, tremor, and weight), behavior (exploration, freezing, and rearing), and neurologic function (grip strength, motor coordination and balance, and pain sensitivity) were assessed. Abnormalities were noted in the pilot experiment at 5 and 11 months after infection and in the initial cohort at 11 to 16 months after infection.
Neurologic and behavioral findings in SPF SW female mice infected at 7 months of age and tested at 12 months of age
Figure 1 Appearance of eleven-month-old Specific Pathogen Free (SPF) mice that are uninfected and chronically infected with T. gondii. (A) Eleven month old uninfected Swiss Webster mouse. (B) Eleven month old infected female mouse from the same SPF colony ten (more ...)
Figure 2 Behavioral and Neurologic Data of eleven-month-old Specific Pathogen Free (SPF) mice that are uninfected and chronically infected with T. gondii. (A) Number of mice with abnormal behavior or neurologic findings. (B) Weight of uninfected and chronically (more ...)
In Figure , the normal, sleek appearance of the uninfected control mouse (Figure ) contrasts with the ruffled fur and tail wound of the infected mouse in Figure and the tilted posture of the infected mouse in Figure . The number of mice with abnormal findings in various aspects of their appearance are shown in Figure . With the exception of stereotyped behavior, palpebral closure, lacrimation, and salivation, the difference between infected and uninfected mice in all of the categories (grooming, body position, piloerection, tail wounds, and tremor) was statistically significant (Table , Figure ).
In this initial cohort, chronically infected mice weighed less than uninfected mice at both 11 and 16 months after infection (Figure ; p = 0.04, p = 0.007). In addition, the infected mice lost weight over time while the uninfected mice gained weight. Locomotion including transfer arousal and gait were both significantly different in infected mice than in uninfected mice (Table ; p = 0.035, p = 0.034). A decrease in autonomic nervous system function of infected mice, measured by increased urination and defecation during brief periods of handling, was also statistically significant when compared to uninfected mice (Table ; p < 0.001). Exploratory movements including sniffs, rears, movement to the center of an open field were significantly less and latency to move significantly greater in infected mice (Figure ; p = 0.0055, p = 0.016, p = 0.017, p = 0.021) and only infected mice exhibited freezing behavior, although it did not achieve statistical significance.
Measures of motor and sensorimotor function also were impaired in infected mice (Figure ). Infected mice displayed a statistically significant increase in the time it took to remove/attempt to remove a paperclip attached to their tail (indicating decreased pain sensitivity as a sensorimotor measure; p = 0.003) and a statistically significant decrease in the length of time that both grip strength and balance were maintained (p = 0.005; p = 0.01; please also see Movie, Additional file 1
in on-line supplement).
Kinetics of development of behavioral and neurologic abnormalities in two additional studies (replicates of each other)
To characterize the kinetics of development of these findings and to make certain a subset of our initial observations were reproducible, two additional experiments with 5 infected mice and 5 control mice in each experiment were performed. In these replicate studies, appearance, gait, exploratory behavior, and pain sensitivity (paperclip test) were evaluated. In both of the replicate experiments, abnormalities in appearance, similar to those found in the initial cohort, were found in some of the infected mice as early as 3 months post infection, the initial time observations were made. By 5 months after infection, four of the ten infected mice in the combined replicate studies had died, two had pronounced gait abnormalities involving their lower extremities, and all but one of the remaining mice had more subtle abnormalities in gait. These latter mice moved their lower extremities more slowly and less facilely and had diminished exploratory behavior when placed in a novel, open environment. One of the three mice with the subtler gait abnormalities had a posture where it was tilted to one side. At the time of the initial observations (3 months post infection), infected mice were taking longer, on average, to notice the paper clip attached to their tail, although this did not reach statistical significance (14.9 ± 13.0 vs. 6.4 ± 8.7 seconds in controls, p = 0.39). At five months, the difference was larger and statistically significant (20.5 ± 11.1 vs. 8.4 ± 7.0 seconds, p = 0.039). No tail lesions, which were noted in the initial cohort, were seen in any of these ten mice before the experiment was terminated.
Brain MRIs of chronically infected mice have mild to moderate ventricular dilatation
To determine whether noninvasive neuroimaging could identify any abnormalities and if so their anatomic distribution, brain MRIs were performed for 6 chronically infected mice (8 months after infection [n = 5] or 12 months after infection in the case of the pilot [n = 1]) and 3 control mice. Representative frames from the MRIs are in Figure . T1 weighted images showed no clear abnormalities between the uninfected and infected mice. T2 weighted images (Figure ) for the control, uninfected mice revealed no abnormalities other than very slight lateral ventricular dilatation. Similar MRI images of the brains of the infected mice, however, showed no (n = 1), mild (n = 1) and moderate (n = 4) lateral ventricular dilatation, which can be seen in the lateral ventricles approximately 0 to 1 mm caudal to bregma when compared to the MRIs from the uninfected mice. Quantitation of the differences in ventricular size at approximately 1 mm caudal to bregma revealed smaller ventricles in the uninfected as compared with the infected mice, which was statistically significant (p = 0.03). In addition, the areas adjacent to the aqueduct of Sylvius had enlargement of the ventricles and periventricular and periaqueductal changes (Figure ). At approximately 6 mm caudal to bregma, the difference in ventricular size between the uninfected and infected mice also was statistically significant (p = 0.002). No parenchymal abnormalities were noted in two of the infected mice, and asymmetry of uptake of contrast in the cortex in the T2 weighted images was noted in the other four infected mice.
Figure 3 MRI findings in chronically infected mice and in uninfected mice. T2 weighted MRIs from uninfected mice (U1-3), chronically infected mice (I1-5), and one infected mouse studied in a pilot experiment at a different time (P1). MRIs were obtained when mice (more ...)
Correlation of brain weight and neurologic findings
Studies of brain weight and correlation with behavioral and neurologic studies were performed to examine whether chronic T. gondii causes loss of brain parenchyma and neuronal cells and whether behavioral and neurologic abnormalities correlated. Such neuronal cell loss was suggested by the brain MRI studies and the increase of GFAP message in the full genome microarrays which reflected neuronal cell injury (see below). In the first behavioral and neurologic experiment, brain weight was not measured. In the second and third replicate experiments described earlier, the mean ± sd [range] of brain weight for infected and control groups were 0.42 ± 0.04 [.385–.466] g and 0.46 ± 0.03 [0.427–0.510] g, respectively (p = 0.065). Further, the infected mice that had more obvious gait abnormalities had the smallest brains. In these experiments a score for abnormal neurologic function and movement pattern was defined as follows: normal gait and exploratory pattern was scored as 0, mild incoordination of gait was scored a 1 or 2, moderate incoordination was scored 3–7 and severe disability in moving one or both hind legs was scored 8–10, with the higher number the most severe. All of the uninfected mice had a score of 0. The uninfected mice quickly moved from the center of the open field to the perimeter usually moving around the perimeter in one direction exploring it. There were no neurologic abnormalities and no association of body size with brain weight present in the 10 uninfected mice (r = -0.024, 95% CI (-0.64, 0.62), p = 0.95). In contrast, all but one of the infected mice moved much less and had a less organized movement pattern around the perimeter of the testing area, with greater severity of that pattern associated with abnormal movement of the hind extremities. The correlation coefficient for the association of abnormal neurologic examination and movement pattern (i.e., higher score as described above) with diminished brain weight in infected mice was -0.53 (95% CI (-0.81, -0.05), p = 0.035).
Microarrays reflect inflammation and increased expression of CD36 and PD1L, GFAP, ubiquitin ligase, and C1q
To determine molecular mechanisms whereby T. gondii
alters brain cell functions and reflects the pathogenic process, full genome microarrays were performed using the MEEBO array containing ~36000 probes representing ~25000 genes. All the probes that were significantly differentially expressed are summarized and listed individually in Table . There were 326 significant probes (corresponding to 311 different genes) with an adjusted P-value less than 0.01 and a posterior log odds of differential gene expression greater than 2 (i.e. a posterior probability greater than 0.88). All these genes showed greater expression in the chronically infected brains and many are associated with the immune response (Table [87
]). There were no significantly downregulated genes. The microarray results are consistent with an inflammatory process involving immunoglobulin and B cells and interferon gamma production. In addition, there was increased expression of the Suppression of Cytokine Signaling (SOCS), CD36, and PD-1L genes and others including C1q. GFAP expression reflects astrocyte response to neuronal cell injury. Ubiquitin ligase expression is increased, likely reflecting effects on host cell protein processing.
Genes upregulated in the brains of mice infected with T. gondii for a year
Histopathologic abnormalities and immunohistochemistry
To better understand the pathologic processes that caused the abnormal behavior and neurologic function described earlier, histopathology of brain and special immuohistochemistry studies focusing on anatomic brain regions also were performed. Consistent with some of the earlier studies of non-SPF mice [7
], histopathological analyses revealed brain abnormalities in the infected SPF mice (Figure ). These findings included mild to moderate diffuse parenchymal infiltrates of inflammatory cells (predominantly lymphocytes) and with the appearance of microglia (Figure ). Collections of lymphocytes and plasma cells appeared around blood vessels of different sizes and in the leptomeninges (Figure ). Focal calcifications were also observed, suggesting a previously healed inflammatory process. Our pathological findings in the context of this chronic infection included rare encysted bradyzoites, the parasite's latent life stage (Figure , Table ). Cysts located within neurons were not associated with an inflammatory response (Figure arrow). Solitary cysts in the brain parenchyma usually were remote from inflammation and calcifications but were occasionally adjacent to, but separate from, perivascular and intra-parenchymal inflammation (Figures arrow).
Figure 4 Inflammation in the brain during chronic Toxoplasma infection with only a few bradyzoites and cysts. (A) Occasional cyst with bradyzoites a short distance from a vessel (arrow), × 250. (B) Medium power view showing perivascular cuffing, × (more ...)
Histopathology in brain regions of uninfected and infected mice
In the brain, CD4+ T cells, CD8+ T cells, plasmacytoid B cells, and activated microglial cells formed prominent perivascular infiltrates (cuffs around vessels of all sizes) (Figures and ) and diffuse parenchymal infiltrates (Figure ) which were not present in micrographs of tissues concomitantly prepared from control mice. In chronically infected mice, the individual plasma cells around vessels were identified by the presence of the immunoglobulin within the cytoplasm (Figures ). In our chronically infected, SPF mice there were rare microglial nodules but no areas of necrosis as seen in immune-compromised persons [89
], mice with the C57BL/6J genetic background [36
], or various other murine models of immune-deficiencies. In sharp contrast to areas of necrosis in genetically susceptible rodents and immune-compromised persons, manifestations of the infection we observed are associated with substantial chronic inflammatory processes without extracellular organisms.
Figure 5 T cells and microglia in brains of chronically infected mice. Representative images of one of five infected mice. T lymphocytes were present in nodules and in the perivascular spaces in frontal cortex and diencephalons. CD4 T lymphocytes and microglia (more ...)
Sections of brains from chronically infected animals and controls also were studied with trichrome, Bodian, Bielshowsky, Luxol blue, and Nissl stains as well as with immunohistochemical stains for neurofilament and amyloid precursor protein to examine for evidence of neuronal loss, demyelination, axonal damage or widespread microglial activation. However, while the brains of chronically infected mice were smaller than controls, despite the presence of focal areas of chronic inflammation, it was not possible to demonstrate neuronal loss, axonal injury nor extensive demyelination.
As also demonstrated by Ferguson et al in separate studies [50
], immunostaining only very rarely identified bradyzoites outside cysts (Figure ) and only one tachyzoite was identified in all the sections examined from many mice (Figure ). However, this confirmed the technique was suitable for the identification of even low numbers of tachyzoites and bradyzoites. (Figures ). The presence of very few extracellular parasites contrasted with the robust immune response in the brain parenchyma, leptomeninges and around blood vessels.
Anatomic distribution of lesions and parasite burden demonstrate different areas of predominance of cysts and calcifications
To better understand the basis for the broad range of behavioral and neurologic abnormalities observed, distribution of lesions and parasite burden in various anatomic areas was characterized (Tables , , and ). Inflammation and parasite burden were greatest in diencephalon (mean ± SD, median, range: 6.4 ± 3.9, 6, 1–13), then cortex (5.6 ± 3.2, 4.5, 2–12), and then hippocampus (4.9 ± 2.4, 4.5, 3–10). The cerebellum showed less parenchymal inflammation, but had perivascular inflammation and calcifications (4.1 ± 1.1, 4, 3–6). The contrast in magnitude of pathology and cyst number in the specific regions of the brain is shown in Table , . However, only some of these differences (cyst number and calcifications) reached statistical significance (p < 0.01 and p = 0.02, respectively).
Comparisons (using Wilcoxon rank-sum test) of infected and uninfected mice
Comparisons of the four different brain regions for infected mice only
Correlations of region of brain involved and behavioral and neurologic findings
Prominent perihippocampal and hippocampal perivascular inflammation
There is an association of hippocampal abnormalities with a number of neurological diseases of humans including Alzheimer's disease, depression, and schizophrenia [56
]. Thus, it is especially noteworthy that in the chronically infected mice there is an area of pronounced inflammation in the leptomeninges contiguous to the hippocampus, particularly around blood vessels (Figure ). This was present in all of the eleven chronically infected mice that survived to the end of the studies in the second and the third replicate experiments. It was present in those that had Magnetic Resonance Imaging (MRI), in which tissues were available and this was specifically examined. It was absent in all the thirteen uninfected controls. This process was noted where the posterior cerebral artery bifurcated (Figure circled) and adjacent blood vessels in the leptomeninges traversed the area next to the hippocampus (Figure arrows). The inflammation extended along the blood vessels as they penetrated into the hippocampus (Figure , hippocampus marked by arrow). The inflammation was pronounced at the most posterior part of this area but also accompanied vessels continuing anteriorly to the areas above the third ventricle and into the blood supply of the basal ganglia. (Figure right panels provide detail of the pathologic process and show the prominence of this peri-hippocampal process and the absence of the process in the leptomeninges overlying the cortex in this micrograph.) The magnitude of the inflammatory process was comparable to other areas of inflammation in the same mouse, but this region was uniformly involved in each of the eleven mice examined. This more prominent inflammatory process is in the area of the brain associated with short-term memory and spatial orientation.
Figure 6 Perivascular inflammatory infiltrates in vessels that supply the hippocampus (circle) adjacent to the hippocampus (labeled H) and in vessels contiguous to and in the hippocampus (arrows) and at the base of the brain. No such inflammatory cells are seen (more ...)
Neurologic findings and histopathology correlate
In the initial, first experiments in which very detailed behavioral assessments were made, there also was a significant correlation between the degree of behavioral/neurological abnormalities and magnitude of the parasite burden or inflammatory process (r = 0.87, 95% CI (0.59, 0.96), p = 0.0002; N = 6 infected and 6 uninfected control mice). A correlation of behavioral and neurologic findings with regions of histopathology is listed in Table . The presence of calcifications, especially in the cerebellum, without current inflammation suggested that there was an earlier inflammatory process with necrosis that had undergone dystrophic calcification. Lesions along the distribution of the motor pathway and the spinocerebellar tract could also account for gait and tail abnormalities. In the second and third replicate experiments, the infected mice with the most abnormal gait had the most severe brain inflammation and the smallest brains, and a mouse with abnormalities of both legs had substantial inflammatory infiltrate in the corticospinal tract.
Prolonged treatment with sulfadiazine does not eliminate pathology
A separate group of similar SPF, chronically infected, female SW mice were treated with sulfadiazine or left as untreated, chronically infected, matched controls to better understand whether conversion of bradyzoites egressing from ruptured cysts into tachyzoites might elicit the inflammatory response we observed. Sulfadiazine, which is a competitive analogue of PABA and inhibits tachyzoite growth, but not encysted bradyzoites, does not eliminate all T. gondii
parasites from congenitally infected or immune-compromised persons [90
]. Sulfadiazine treatment administered to our chronically infected mice for 4 months did not significantly modify pole balance (16.4 ± 12.9, range = 0–29 versus 23.6 ± 23.8, range = 2–60; p = 0.68) or grip test results (10.8 ± 5.4, range = 5–19 versus 9.6 ± 11.3, range = 1–29; p = 0.40) or the inflammation and perivascular cuffing in the brain. Treatment with sulfadiazine did reduce, but did not eliminate, the number of cysts (4.8 ± 0.8, range = 4–6 versus 8.0 ± 1.0, range = 7–9; N = 5 mice per group, p < 0.01). These values are for number of cysts in 50 microliters of half a brain, which was homogenized and suspended in 2 ml. Thus, numbers for the whole brain were these numbers of cysts multiplied by 80.
Heart and large vessels and levels of lipoproteins and inflammatory mediator SAA do not reflect a systemic inflammatory process
To better understand whether the inflammatory process in brain was part of a systemic inflammatory process in chronically infected mice, heart and large vessels, levels of lipoproteins, and the inflammatory mediator SAA were measured. There was no inflammation in the myocardium, and atheromatous plaques were not present in large vessels of mice that had the abnormal neurological findings and brain histopathology (Table , ) in the first experiment (data not shown). Consistent with the absence of this latter finding in our mice, plasma levels of lipoproteins were not altered and assays for the inflammatory mediator SAA from our chronically infected mice showed that there was essentially no SAA in the plasma samples from either control or chronically infected mice (data not shown).
Effect of antibody to PD-1L on amount of inflammatory infiltrate and number of cysts
Because there was increased expression of PD-1L in whole genome microarrays, and this is a ligand that allows persistent Lymphochoriomeningitis virus brain infection by limiting activity of T cells, hamster antibody to PD-1L or isotype-control was administered every 7 days for 21 days. There were three mice tested in each of the following groups: no antibody, isotype-control antibody, and antibody to the PD-1L ligand. Hamster antibody to PD-1L, which has been demonstrated in other studies to abrogate murine PD-1L function, did not significantly decrease the number of cysts (p = 0.12). The median number of cysts were 13, 6, and 5 in the no antibody, isotype-control antibody, and PD-1L antibody groups, respectively. Additionally, inflammation was present in all the groups (p = 0.56). In the mice that received the anti-PD-1L there appeared to be an increased amount of inflammatory infiltrate relative to the numbers of cysts.
Administration of isolated parasites produces the same pathology
To address whether infection with concomitant pathogens or parasites administered together with inoculation of brain tissue caused the brain pathology, histopathology of brain from SPF mice chronically infected with Me49 strain (clonal type II) parasites, inoculated without brain tissue, was also studied. The pathology in these mice infected with bradyzoites from isolated cysts (Figure ) was similar to that described above.
Figure 7 (A-C) Similar histopathology with perivascular inflammation, isolated cyst ×40, and cluster of microglia in a mouse that is chronically infected, initially infected with parasites without accompanying brain. A ×40; B ×250; C ×20. (more ...)
Genetically resistant BALB/c mice also have perivascular and leptomeningeal inflammation, but there is much less
To determine whether a genetically resistant strain of mouse would have the same type of pathology when chronically infected, chronic infection of BALB/c mice, a strain that is genetically resistant to acute infection and toxoplasmic encephalitis, was also studied. In SPF BALB/c mice infected with clonal type II Me49 parasites for 6 months (N = 5 infected, 3 controls), there were only very rare cysts seen but there was a small amount of perivascular accumulation of inflammatory cells in 4 of the 5 infected mice and leptomeningeal inflammation that was not seen in the controls. These results were similar to studies of brains of BALB/c mice infected for 60 days. Figure is a representative example of this pathology figure is a higher power view of this area of brain. All analyses of tissues of these mice were performed by a pathologist without knowledge of the infection status of the mice from which the tissue was derived.
Subacute infection in mice with knockout of IL-4, IL-6, IL-13 or NRAMP elicits the same brain histopathology
Because IL-4, IL-6, IL-13 and NRAMP are very important in immunity to toxoplasmosis and could play a pathogenic role in the inflammatory process we observed, mice without IL-4, IL-6, IL-13, or Nramp, infected for shorter times, were also studied to determine whether these cytokines or immune processes were necessary and sufficient for the perivascular cuffing and meningeal and parenchymal abnormalities we had observed. These mice also had prominent perivascular cuffing and parenchymal infiltrates, in a distribution similar to that in the outbred mice (FR, CWR, data not shown) indicating that these cytokines and NRAMP were neither necessary nor sufficient to cause the pathology we observed.
Brain histopathology of mice that had MRIs
To determine whether the type of histopathology we observed would be reflected in a conventional MRI, immediately following the MRIs the brain was removed and fixed in formalin for subsequent histopathologic analysis. The histopathological changes in brain were similar to those described above for the infected mice showing mild to moderate parenchymal inflammation, perivascular cuffing with inflammatory cells, and leptomeningeal inflammation with scattered cysts without inflammation. The uninfected age-matched controls housed in the same colony for 13 months had no brain pathology (p = 0.02 for comparison to infected mice). Figure shows the brain histopathology of a representative mouse with only slight ventricular dilatation.
Electron microscopy and immunostaining of the intact and ruptured tissue cysts
The intact cysts were located in neurons identified by the presence of synapses between the host neuron and adjacent neurons (Figure ). On extremely rare occasions it was possible to observe tissue cyst rupture, which was associated with loss of the host cell (Figure , inset). It was found that mononuclear cells were surrounding the cyst and invading through the ruptured cyst wall. The monocytes were attacking and engulfing the bradyzoites (Figure ). The bradyzoites were destroyed before they could convert to tachyzoites.
Figure 8 (A-B) Details of the periphery of tissue cysts from chronically infected mice identifying the host cells (HC) as neurons due to the formation of synapses (arrows). Based on the structure of the synapse the cysts appear to be within a neuronal dendrite. (more ...)