Long-term clinically relevant rodent model of methotrexate-induced cognitive impairment

Abstract

Background

With the enhanced use of chemotherapy and the advent of increased patient survival rates, there are an increasing number of cancer survivors living with chemotherapy-induced cognitive impairment. A growing number of clinical studies have brought to light the association of agents like methotrexate in generating these neurological sequelae, although mechanisms remain unclear.

Methods

Here, we use a clinically relevant regimen of several cycles of methotrexate and leucovorin rescue to develop a model of chemotherapy-induced cognitive impairment, and investigate the in vivo long-term (16 mo) impact of high-dose systemic methotrexate on white matter cellular dynamics as assessed by stereology, animal behavior, and diffusion tensor imaging.

Results

Our results indicate that at 6 and 16 months post-chemotherapy, methotrexate-treated rats exhibit a significant and permanent decrease in the number of oligodendrocytes and their progenitors in the white matter, in corpus callosum volumes, and myelin basic protein. These findings are associated with mostly delayed deficits in performance on Morris Water Maze and Novel Object Recognition tasks. Diffusion tensor imaging demonstrates significantly decreased fractional anisotropy values in the callosum genu, body, and splenium, as well as previously unassessed areas like the fimbria. Interestingly, these white matter changes are preceded by an earlier, transient decrement in white matter microglia at 3 months, and hippocampal neural progenitors at 3 and 6 months.

Conclusion

These results demonstrate a significant negative impact of methotrexate on the oligodendrocyte compartment and white matter, associated with cognitive impairment. The data also support the use of diffusion tensor imaging in monitoring white matter integrity in this context.

Key Points.

1. Methotrexate permanently suppresses oligodendrocytes.

2. High-dose systemic methotrexate leads to cognitive deficit.

3. Diffusion tensor imaging reveals methotrexate-related alterations in the white matter tract.

Importance of the Study.

High-dose methotrexate permanently suppresses the oligodendrocyte populations in the brain and leads to cognitive impairment. White matter disruption is detectable by diffusion tensor imaging.

Advances in cancer treatments over the last decades have led to improved outcomes and survival rates in cancer patients.¹ However, the increase in survival of cancer patients has not always translated into an increased quality of life. In fact, while there are expected to be roughly 70 million cancer survivors in the world by the year 2020,^2,3 a significant proportion are expected to develop neurological dysfunction related to chemotherapy in the years following treatment.^4,5 These impairments, which are well documented and widespread across different chemotherapy regimens, include problems with memory, attention, speed of processing, executive function, thought process, and mood disorders.^2–4 The collection of these effects has been termed chemotherapy-induced cognitive impairment (CICI), or colloquially, “chemobrain.” ²

A growing number of preclinical studies designed to investigate the mechanisms of CICI have brought to light the role of drugs like methotrexate (MTX) in generating these neurological sequelae.^6–8 Proposed mechanisms are diverse, but studies have revealed an impact on apoptosis, blood supply, CSF composition, and inflammation, among others.^7,9 The chemotherapeutic agent, MTX, deserves further study in particular due to its association with CICI^10,11 and its use in multiple oncologic regimens in high doses.

High-dose MTX is a component of modern treatment protocols for acute lymphoblastic leukemia (ALL),¹² systemic non-Hodgkin’s lymphoma,¹³ pediatric osteosarcomas,¹⁴ and primary central nervous system lymphoma (PCNSL).¹⁵ In fact, MTX remains the mainstay of PCNSL therapy as a single agent or in combinations.¹⁵ MTX is also an important drug for the management of leptomeningeal metastases whereby it is administered intrathecally.¹⁶ A recent prospective brain MRI study in children who underwent MTX induction for ALL revealed an association with persistent, long-term leukoencephalopathy,¹⁰ demonstrating that the effects of CICI may have some correlate with structural white matter injury.

While the effects of MTX in animal models have largely been demonstrated in gray matter areas like the hippocampus,^17–20 the potential role of MTX on white matter tracts and oligodendrocyte progenitor cell (OPC) populations has received less attention. These cycling cells are particularly vulnerable to anti-metabolite activity by MTX. To date, only a single in vivo study has investigated the influence of MTX on white matter oligodendroglia and myelination in a juvenile animal model of CICI, with no animal studies focusing on the long-term effects of this chemotherapy regimen (ie, past 6 mo treatment). Here we investigate the long-term impact of high-dose systemic MTX on brain structure, with particular attention to oligodendroglia and myelination, hippocampal neurogenesis, and microglia.

Materials and Methods

Rat Maintenance

Male and female Sprague-Dawley rats aged 3 weeks (Charles River Laboratories) were randomized into 2 groups, control (n = 18) and MTX rats (n = 17). All procedures were performed in accordance with federal guidelines and supervised by the Institutional Animal Care and Use Committee.

Chemotherapy

We aimed to develop a treatment regimen that is representative of common MTX treatment regimens in children. Rats belonging to the treatment group (aged 3 wk) were given MTX via intraperitoneal injection every week at a dose of 200 mg/kg/week for a total of 4 weeks, and a final dose of 800 mg/kg (Fig. 1). No adverse effects from MTX therapy (including weight loss, diarrhea, or death) were observed during the treatment period. Leucovorin rescue was administered using standard guidelines to prevent MTX-induced acute neurotoxicity (for detailed regimen and rationale, see Supplement).

Fig. 1.

Experimental design. Experimental setup for juvenile MTX-exposure paradigm. Sprague-Dawley rats aged 3 weeks were given weekly doses of intraperitoneal MTX, followed by leucovorin rescue 1 and 2 days after each MTX dose. Experimental endpoints were set at 1.5, 3, 6, and 16 months following chemotherapy, at which time behavioral testing (Morris Water Maze and Novel Object Recognition) and immunohistochemistry were performed. Brain regions analyzed stereologically included the corpus callosum and hippocampus (gray shaded regions). DTI was performed at 16 months.

Novel Object Recognition

We used a previously published, modified version of the Novel Object Recognition task to test for attention and short-term memory (see Supplement).^9,21

Morris Water Maze

The Morris Water Maze (MWM) test was performed as previously described to assess for long-term memory function in the hippocampus²² (see Supplement).

Perfusion and Immunohistochemistry

Animals were sacrificed at 1.5, 3, 6, and 16 months for immunohistochemistry following standard protocols (see Supplement) and stained for Ki67, oligodendrocyte transcription factor 2 (Olig2), ionized calcium binding adaptor molecule 1 (Iba1), or myelin basic protein (MBP).

Stereology

Stereology was performed using the optical fractionator probe in Stereo Investigator 2017 (MBF Bioscience) by a blinded investigator. For details, see Supplement.

Diffusion Tensor Imaging

Ex vivo MRI scan was performed on a vertical Bruker 11.7T 89 mm bore magnet. Regions of interest were delineated manually and were used to calculate corresponding regional fractional anisotropy (FA), axial diffusivity (AD), radial diffusivity (RD), and mean diffusivity (MD) (for details, see Supplement).²³

Statistical Analysis

Results are expressed as the mean ± SEM. Statistical analysis was performed using GraphPad Prism v7. All statistical analyses were performed by two-tailed Student’s t-tests. P ≤ 0.05 was considered statistically significant. All statistical analyses were blinded.

Results

Methotrexate Causes Long-Term Disruption of White Matter

We conducted a quantitative analysis of the number of cells expressing Olig2 (an established marker of oligodendroglia²⁴) in the corpus callosum, using a stereological approach. Stereology is an unbiased approach that uses methods to ensure rigorous quantitative analysis of different parameters, in this case, the optical fractionator probe to estimate cell numbers. We identified a steady decrease in the number of Olig2+ cells in the corpus callosum of MTX-exposed rats when compared with controls, reaching significance at 6 and 16 months (decrease of 64% and 42%; P = 0.0002, P = 0.0463, respectively) (Fig. 2A, B). Within the control group, there was a significant expansion of Olig2+ cells by 45% from 1.5 to 6 months (P = 0.0232), followed by a significant decrease of 47% from 6 to 16 months (P = 0.0022) (Fig. 2B). This may represent the natural expansion of the OPC population in the maturing brain, followed by aging-related losses. There was no significant difference among timepoints in the MTX-exposed rats, as Olig2+ cell populations remained consistently low post-chemotherapy (Fig. 2B).

Fig. 2.

Methotrexate causes long-term disruption of OPC populations and myelination in white matter. (A) Representative images of corpus callosum immunohistochemistry stains for Olig2+/4′,6′-diamidino-2-phenylindole (DAPI) from control or MTX-exposed rats at 1.5, 3, 6, and 16 months. (B) Olig2+ cells in the corpus callosum are significantly decreased in MTX-exposed rats at 6 and 16 months. (C) Representative MBP+ immunohistochemistry of the corpus callosum. (D) MTX-exposed rats have significantly decreased mean MBP immunofluorescence intensities of the corpus callosum at both 6 and 16 months. (E) Corpus callosum volumes are decreased in MTX-exposed rats at 6 and 16 months. Scale bars = 100 µm. 1.5 months (n = 2 control; n = 3 MTX); 3 months (n = 2 control; n = 3 MTX); 6 months (n = 4 control; n = 4 MTX), 16 months (n = 4 control; n = 4 MTX). Data are shown as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Unpaired two-tailed Student’s t-test.

We also evaluated expression of MBP, a marker of mature oligodendrocytes, in the corpus callosum by immunofluorescence. The data showed significantly lower MBP in the MTX-exposed rats at both 6- and 16-month timepoints compared with controls (decrease of 25% and 15%, respectively; P = 0.0012, P = 0.0045, respectively), indicating a deficit in the ability of MTX-exposed rats to form or maintain mature myelin, possibly due to the loss of OPCs (Fig. 2C, D). In the control group, there was a significant increase in MBP mean intensity from 1.5 to 3 months (P = 0.0316) and 3 to 6 months (P = 0.0174), but not from 6 to 16 months (P = 0.1502) (Fig. 2D). Within the MTX-exposed rats, this peak in MBP mean intensity was suppressed, with differences not becoming apparent until the 3- versus 6-month timepoints (P = 0.0395) (Fig. 2D).

Along with a long-term decrease in Olig2⁺ expressing cells, the corpus callosum volumes, also measured stereologically, revealed significantly lower volumes in MTX-exposed rats at 6- and 16 months when compared with controls (decrease of 24%, 14%; P = 0.0007; P = 0.0212, respectively) (Fig. 2E). This difference was not apparent at 1.5- or 3 months in controls versus MTX-exposed rats (P = 0.1137, P = 0.2556, respectively) (Fig. 2E). Taken together, these data suggest a significant and sustained injury to the Olig2+ expressing cells, as well as to the mature myelinating oligodendrocytes, consequent to MTX exposure.

Total Microglia Cell Populations Are Suppressed in Response to Methotrexate

To assess the impact of MTX on the microglial cell populations in white matter, stereological analysis was conducted in the corpus callosum using Iba1 as a marker of microglia.^9,25 At 3 months, microglia decreased significantly in MTX-exposed rats by 66% compared with controls (P = 0.0191) (Fig. 3A, B). Interestingly, the microglia in MTX-exposed rats began to recover after 3 months, gradually increasing significantly from 3 to 16 months (P = 0.0104) (Fig. 3B). Analysis of microglia in age-matched controls indicated a significant decrease between 3 and 6 months (P = 0.0089) of 65%, followed by a significant increase in the aging rat between 6 and 16 months (P = 0.0191) of 154% (Fig. 3B). There was no significant difference in microglia between control and MTX rats at other timepoints.

Fig. 3.

Methotrexate causes long-term suppression of microglia in the white matter and neurogenesis in the hippocampus. (A) Representative corpus callosum immunohistochemistry (IHC) for Iba1 from control or MTX-exposed rats. (B) Iba1+ microglia are significantly decreased in MTX-exposed rats at 3 months compared with controls. (C) Representative Ki67+/4′,6′-diamidino-2-phenylindole (DAPI) IHC in the hippocampal dentate gyrus in control or MTX-exposed rats at 1.5, 3, 6, and 16 months. (D) Ki67 is significantly lower in MTX-exposed rats at 3 and 6 months. Scale bars, Iba1 = 100 µm, Ki67/DAPI = 50 µm. 1.5 months (n = 2 control; n = 3 MTX); 3 months (n = 2 control; n = 3 MTX); 6 months (n = 4 control; n = 4 MTX), 16 months (n = 4 control; n = 4 MTX). Data are shown as mean ± SEM. n.s. = P > 0.05, *P < 0.05; **P< 0.01; ***P < 0.001; ****P < 0.0001 by unpaired two-tailed Student’s t-test.

Methotrexate Causes Long-Term Suppression of Neurogenesis in the Hippocampus

In view of the impact of MTX on cell division, we quantified the number of proliferating neuroblasts in the hippocampus via stereology. The number of Ki67+ cells was significantly lower in the MTX-exposed rats compared with controls at both 3 and 6 months (decrease of 60% and 66%; P = 0.0322, P = 0.0156, respectively) (Fig. 3C, D). This difference between control and MTX-exposed rats was trending but not statistically significant at 16 months (P = 0.1878), as there were very few Ki67+ cells in either condition at this timepoint (265 ± 93.14 vs 112.5 ± 43.08) (Fig. 3D).

Both control and MTX-exposed rats experienced a decline in the population of Ki67+ cells in the dentate gyrus over time (Fig. 3D). However, this expected decline occurred earlier in the MTX-exposed rats, with a significant decrease in Ki67+ cells from 1.5 to 3 months (P < 0.0001) and 3 to 6 months (P = 0.0069), suggestive of a significant injury early post MTX exposure, with an inability to recover over the lifetime of the rat. Neurogenesis in the dentate of the normal rats seemed to peak in the first 3 months of life, with a substantial decline at 6 months (P = 0.0101, compared with the 3-mo timepoint) and beyond (P = 0.0046 at 16 months, compared with 6 mo) (Fig. 3D).

Persistent Neurocognitive Deficits Following Methotrexate Exposure in Rats

We utilized 2 different behavioral paradigms to test rats at all timepoints. The Novel Object Recognition task was used to assess for attention and short-term memory.²¹ During the testing trials at 1.5, 3, and 6 months, rats from control and experimental groups were able to significantly discriminate between the novel object and the familiar object (Fig. 4A, B). However, at 16 months, MTX rats were no longer able to discriminate between novel and familiar objects (P = 0.8087) (Fig. 4A). This is in contrast to control rats, which maintained an ability to discriminate at 16 months (P = 0.0402) (Fig. 4A). These data suggest a delayed disruption of short-term memory and attention in MTX-exposed rats.

Fig. 4.

Novel Object Recognition task reveals development of short-term memory and attention deficits in MTX-exposed rats. MTX-exposed rats perform worse on the Novel Object Recognition task at 16 months, demonstrating a significant inability to discriminate between novel and familiar objects and thus a deficit in short-term working memory and attention. Control rats are not affected. (B) Schematic of the experimental setup for the training phase of behavioral testing (2 familiar objects) and the testing phase, where one familiar object has now been replaced with a novel object. 1.5 months (n = 2 control; n = 3 MTX); 3 months (n = 2 control; n = 3 MTX); 6 months (n = 4 control; n = 4 MTX), 16 months (n = 4 control; n = 4 MTX). Data are shown as mean ± SEM. n.s. = P > 0.05, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by unpaired two-tailed Student’s t-test.

The MWM—which correlates with spatial orientation, learning, and hippocampal plasticity—was used to assess for long-term memory²² (Fig. 5). On the test trial day at 1.5, 3, 6, and 16 months, control rats spent significantly more time swimming in the target quadrant than any other quadrant, indicating intact long-term reference memory (Fig. 5A, B). In contrast, MTX rats spent significantly more time in the target quadrant at 1.5 months, but then at 3, 6, and 16 months they failed to spend more time in the target quadrant compared with ≥1 other quadrants on the test day (Fig. 5A).

Fig. 5.

MTX-exposed rats exhibit deficits in the Morris Water Maze task. (A) MTX-exposed rats spend equivalent amounts of time in the target platform quadrant versus ≥1 other quadrants during the test trial day at 3, 6, and 16 months. Control rats spend significantly more time in the target quadrant at all timepoints. (B) Experimental scheme. On days 1–3, rats underwent 2 training trials with the rescue platform in the same location. On testing day, the rescue platform was removed, and all rats underwent 1 trial with release from the same quadrant. Long-term memory was considered intact if the rat spent more time in the target quadrant than any other quadrant. (C) Heatmap analysis of combined MTX-exposed or control rat testing trials at 16 months. For all animals, the release quadrant was in the Northwest, and the target quadrant was in the Southeast. Closer analysis reveals a more circumferential search pattern in the MTX rats (right) that contrasts the classical spatially oriented search strategy of control rats (left). 1.5 months (n = 2 control; n = 3 MTX); 3 months (n = 2 control; n = 3 MTX); 6 months (n = 4 control; n = 4 MTX), 16 months (n = 4 control; n = 4 MTX). Data shown as mean ± SEM. n.s. = P > 0.05, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by unpaired two-tailed Student’s t-test.

We also performed a heatmap analysis of combined MTX-exposed or control rat testing trials at 16 months. Closer analysis reveals a search pattern of circling in close proximity to the pool walls in the MTX-exposed rats that contrasts with the classic spatially oriented search strategy of control rats (Fig. 5C). Taken together, the behavior data analyses indicate a sustained disruption of learning, long-term memory, and reference memory in MTX-exposed rats that persists out to 16 months.

Diffusion Tensor Imaging Demonstrates Alterations in White Matter Tracts and Highlights New Areas of Methotrexate Damage

Using adaptations of previously established protocols for ex vivo DTI of rat brains,^26,27 we imaged control and MTX-exposed rats at the 16-month timepoint. Seven anatomic regions were selected for analysis: 6 regions were white matter tracts including the anterior commissure, corpus callosum genu (CC_g), corpus callosum body (CC_b), corpus callosum splenium (CC_s), fimbria, and internal capsule (Fig. 6A); the seventh region was the hippocampal dentate gyrus (Fig. 6A). Each anatomic region underwent measurement of FA, MD, AD, and RD (see Materials and Methods).

Fig. 6.

Alterations in specific white matter tracts on DTI. (A) Representative T2 MR images (top) and corresponding directionally encoded color FA maps (bottom) from a 16-month control rat. Shaded regions highlighted in blue represent the specific region of interest analyzed. (B) DTI analysis of select anatomic regions revealed significantly decreased FA values in the white matter tracts in MTX rats vs controls (n = 4 control; n = 4 MTX). anterior commissure (ac), corpus callosum genu (CC_g), corpus callosum body (CC_b), corpus callosum splenium (CC_s), internal capsule (ic), dentate gyrus (dg), fractional anisotropy (FA), radial diffusivity (RD), mean diffusivity (MD), axial diffusivity (AD). Data are shown as mean ± SEM. *P < 0.05; **P < 0.01; two-tailed Student’s t-test; n = 4/group.

Analysis of FA, which is highly sensitive to microstructural damage, revealed significantly lower values in the majority of white matter tracts in MTX-exposed rats. Specifically, FA was significantly lower in the anterior commissure (P = 0.0123), CC_g (P = 0.0114), CC_b (P = 0.0161), CC_s (P = 0.0179), and fimbria (P = 0.0062) of MTX-exposed rats (Fig. 6B). FA was not significantly different among MTX versus controls in the internal capsule or dentate gyrus (Fig. 6B). DTI analysis also revealed increased RD and MD values in MTX-exposed rats, although this finding was not statistically significant. There was no difference in AD in control versus MTX-exposed rats (Fig. 6B).

Discussion

Effects on White Matter

Our data demonstrate that systemic doses of MTX lead to a significant long-term decrease in the number of oligodendroglia in white matter, as well as a decrease in the extent of myelination and associated corpus callosum volume. DTI on MRI revealed evidence of microstructural damage to the white matter in specific tracts, as demonstrated by the decrease in FA. Analysis also revealed a significant, early, and sustained impact on hippocampal neurogenesis beginning at 3 months post-chemotherapy. These findings are associated with an impact on behavioral tests, namely in the MWM, which evaluates processes related to long-term memory and hippocampal plasticity, and the Novel Object Recognition test, which is related to frontal-lobe processes and short-term memory and attention. These behavioral deficits are already established as cognitive sequelae of CICI.^2–4,10

Therefore, it seems that systemic MTX damages the main proliferating cells in the brain, namely the neuroblasts in the dentate gyrus and the proliferating oligodendroglia in the white matter, which is concordant with the known mechanism of activity of this agent. The kinetics of depletion in these populations appear to be different, with an earlier impact on hippocampal neurogenesis. This could be due to a higher rate of proliferation in the dentate gyrus that is potentially more sensitive to the effects of MTX chemotherapy. The subsequent loss of Olig2+ cells likely also leads to a significant deficit in mature oligodendrocytes and an impaired ability to replenish myelin turnover.

Correlating the behavioral abnormalities with histological findings is challenging and requires more extensive assessments, but it is noteworthy that the significant loss of hippocampal neurogenesis seen in the early timepoints did not correlate with an impact on the behavioral tasks. Instead, the more significant behavioral differences between experimental groups occurred later in the animal’s life and several months after the last MTX dose, coinciding with the onset of more significant loss of white matter integrity. Additionally, we demonstrate by DTI that impact on the white matter involves multiple tracts, including the fimbria, which contain the afferent and efferent pathways of the hippocampus. Injury to the hippocampal fibers could contribute to memory disturbances and impairment of spatial orientation, as seen in the MWM results. Concomitant suppression of hippocampal neurogenesis may contribute to these sequelae. The wider involvement of the white matter could also result in impairment of multitasking and decreased attention span and a broader negative impact on cognition. Such abnormalities are commonly reported in patients with CICI and are more challenging to model in rodents.

The Role of Microglia

The role of microglia in a range of CNS disorders is a subject of increasing interest. The only other comprehensive study⁹ to date on the effects of MTX in white matter administered MTX to mice at a dose of 300 mg/kg total (vs 800 mg/kg in the present study), without leucovorin rescue. In that study, microglia numbers increase post MTX, unlike our findings, whereby microglia numbers decrease at 3 months, in comparison to controls. Several differences in methodology may explain the discrepancy: our study uses stereological counting rather than image density, a higher dose of MTX, and leucovorin rescue. Leucovorin is readily converted into folic acid derivatives, and its vitamin function is unaffected by MTX-induced inhibition of dihydrofolate reductase. In the clinical setting, leucovorin rescue is always given following MTX chemotherapy, to prevent the known complication of MTX-related acute neurotoxicity and other systemic effects.^28,29

In the control animals, the number of microglia reaches nadir at 6 months posttreatment followed by a robust increase as the animals reach an advanced age (Fig. 3). These findings in untreated animals are compatible with other investigations, which have shown that Iba1+ microglia populations undergo a significant expansion in the corpus callosum of normal aging rats.²⁵

The data in the MTX-treated rats suggest a relatively suppressed microglia population especially in the early timepoint, such as 3 months. This could be related to a persistent anti-inflammatory impact by MTX. However, the microglia numbers recover later and achieve a peak at 16 months, similar to the normal rats. These kinetics are distinct from those exhibited by the Olig2 cell population and the dividing neuroblasts in the dentate, both of which are permanently suppressed and gradually depleted throughout the life of the MTX animals. This is highly suggestive of different mechanisms underlying the effects of MTX on the diverse cells of the CNS. MTX is also known to be an immunomodulator, thus a more in-depth analysis of the subtypes of microglia and their potential role in myelin turnover and oligodendrocyte population survival would be necessary for our understanding of the role of microglia in CICI.

A Clinically Relevant Model of Chemotherapy-Induced Cognitive Impairment

Here we present a robust rodent model of long-term CICI that is, to the best of our knowledge, the longest study of its kind carried out in rodents to date (16 mo post-MTX), and takes into account factors such as juvenile age and repeated cycles of chemotherapy. While other studies have analyzed the effects of MTX in rodent models (almost entirely in the gray matter), the study period is usually brief (a few days to months post-MTX).^6–8

We implemented a treatment regimen as close as possible to human dosing schedules, which includes repeated cycles of MTX, and leucovorin rescue. Including leucovorin in the regimen ensures that the data obtained are not due to the preventable acute toxicity of MTX. This reasoning, as well as pilot studies in rats demonstrating that high-dose MTX alone can be lethal due to weight loss and diarrhea, and the previous observation that leucovorin itself does not have an observable effect on neurogenesis, justified its use in the present study.¹⁸

Additionally, our regimen is modeled after current treatments for children with ALL^10,30; however, patients often receive far more cycles over a year or longer. It is also important to note that MTX is rarely given as monotherapy; in addition to other chemotherapeutic agents, cranial radiation may frequently be added, particularly in patients with CNS involvement.³¹ This patient group may also receive intrathecal MTX to ensure high CSF concentrations. These additional interventions are known to further increase CNS toxicity, compared with systemic MTX alone.^10,31,32 Thus, our animal model likely represents an underestimation of the CNS toxicities experienced by many patients.

DTI as a Method for Tracking CICI Damage: Benefits as a Noninvasive Strategy in Humans

We present the first time DTI has been utilized to assess for damage from MTX in rodents. We used FA (highly sensitive to microstructural changes in white matter), MD (sensitive to cellularity, edema, and necrosis), AD (normally decreased in axonal degeneration), and RD (increased in white matter demyelination and dysmyelination) as frequently used measures, although FA is most sensitive to white matter damage.^26,27,33 We have shown that white matter alterations, though not immediately obvious on standard MRI sequences, are detectable by DTI analysis. Achieving good resolution is limited by the need to maintain rats under anesthesia for a prolonged period of time. We therefore used a modified ex vivo DTI protocol, a high magnetic field (11.7T), and a long imaging time in order to enhance image quality and resolution (117 µm), which is compatible with currently available human imaging. Ex vivo rodent DTI has previously been validated against in vivo rodent DTI in models of status epilepticus,²⁷ traumatic brain injury,²⁶ and other diseases, but not CICI.

We also evaluated anatomically distinct white matter tracts in order to identify potentially selective vulnerabilities. The DTI findings are consistent with the immunohistological changes observed in white matter tracts of our rodent model, such as suppression of oligodendroglia and deficits in myelin, both detected in the corpus callosum at 6 and 16 months post-MTX. Our analysis revealed significantly decreased FA values indicative of microstructural damage in the corpus callosum, anterior commissure, and fimbria, all of which are involved in hemispheric cross-talk and connections to the limbic system. Interestingly, the internal capsule, which includes the major motor pathways (corticospinal tract), did not show significant alterations, compatible with the lack of primary motor symptoms in rodents and in humans.^10,31

The decreases in FA in the anterior commissure or fimbria are novel findings and warrant further investigation for their possible role in the cognitive sequelae of CICI.⁹ For instance, alterations in the fimbria, which relays afferent and efferent fibers in the hippocampus, may contribute to the long-term memory symptoms experienced by animals and patients, whereby the focus is often placed exclusively on neurogenesis in the dentate gyrus.

The main findings of human studies utilizing DTI to assess CICI have been decreased FA values in white matter tracts.³³ Here we have shown that these observed DTI changes are conserved in a preclinical model of CICI as well, with significantly decreased FA in the abovementioned tracts. This imaging approach offers both a noninvasive assessment of white matter integrity and a potential link between animal and human studies of CICI. DTI could hold promise as a tool not only to assess for damage, but also to track progress of potential reparative strategies.

Therapies to Improve Outcomes from CICI

There are no currently approved FDA therapies for CICI. In vivo studies have looked at the use of microglial inhibitors⁹ or pharmacological modulation such as fluoxetine³⁴ to prevent adverse effects following chemotherapy. Although some of these therapies may hold promise for preventing CICI, they do not address existing deficits. The identification of specific and persistent depletion of OPCs as a key consequence of chemotherapy may represent, upon further validation, an excellent target for cell-based regenerative approaches. Animal models of radiation injury also exhibit loss of oligodendrocyte precursors and disruption of myelination, associated with behavioral and locomotor balance deficits.²¹ Grafts of human OPCs into the irradiated brain were shown in preclinical studies to successfully replenish oligodendrocyte progenitors, reduce loss of myelination, and ameliorate existing behavioral deficits.²¹ Human embryonic stem cell–derived oligodendrocyte progenitors have entered the realm of clinical trials and could represent a potentially successful strategy aimed at repairing white matter injury and improving cognitive deficits in CICI patients.

Limitations

There are potential limitations to the behavioral tasks employed in this study. Additional tests could be performed to provide a more comprehensive perspective on key features of CICI, including evaluation of anxiety and assessment of spatial learning and memory using different mazes (Oasis, Barnes, etc) to minimize confounding parameters. In addition, we performed DTI at the final timepoint of this study, 16 months post-chemotherapy. As DTI techniques and software analysis become more accessible, it would be beneficial to track quantitative parameters over the lifetime of the individual. Finally, further characterization of microglia activation states in response to MTX over time would further expand our understanding of their response to chemotherapy.

Conflict of interest statement. V.T. is co-founder of Blue Rock Therapeutics.

Authorship statement. CB conducted the tissue processing, stereology, behavior, analysis, and manuscript writing. KL performed the animal treatments and behavioral experiments. HCL performed the MRI imaging and analysis. KH addressed microglia questions. SP helped with the analysis and stereology. JP supervised tissue processing, analysis, and stereology. VT oversaw the project and manuscript writing. All authors were involved in manuscript writing, and have read and approved the final version.

Funding

This project was supported by an NIH core grant, P30 CA08748. C.B. was partially supported by a Neurosurgical Research and Education Foundation Medical Student Summer Research Fellowship. S.P. was supported by a Medical Scientist Training Program grant from the National Institute of General Medical Sciences of the National Institutes of Health under the award number T32GM007739 to the Weill Cornell/Rockefeller/Sloan Kettering Tri-Institutional MD-PhD Program.