Stem cell transplantation reverses chemotherapy-induced cognitive dysfunction - PubMed (original) (raw)

Stem cell transplantation reverses chemotherapy-induced cognitive dysfunction

Munjal M Acharya et al. Cancer Res. 2015.

Abstract

The frequent use of chemotherapy to combat a range of malignancies can elicit severe cognitive dysfunction often referred to as "chemobrain," a condition that can persist long after the cessation of treatment in as many as 75% of survivors. Although cognitive health is a critical determinant of therapeutic outcome, chemobrain remains an unmet medical need that adversely affects quality of life in pediatric and adult cancer survivors. Using a rodent model of chemobrain, we showed that chronic cyclophosphamide treatment induced significant performance-based decrements on behavioral tasks designed to interrogate hippocampal and cortical function. Intrahippocampal transplantation of human neural stem cells resolved all cognitive impairments when animals were tested 1 month after the cessation of chemotherapy. In transplanted animals, grafted cells survived (8%) and differentiated along neuronal and astroglial lineages, where improved cognition was associated with reduced neuroinflammation and enhanced host dendritic arborization. Stem cell transplantation significantly reduced the number of activated microglia after cyclophosphamide treatment in the brain. Granule and pyramidal cell neurons within the dentate gyrus and CA1 subfields of the hippocampus exhibited significant reductions in dendritic complexity, spine density, and immature and mature spine types following chemotherapy, adverse effects that were eradicated by stem cell transplantation. Our findings provide the first evidence that cranial transplantation of stem cells can reverse the deleterious effects of chemobrain, through a trophic support mechanism involving the attenuation of neuroinflammation and the preservation host neuronal architecture.

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Figures

Figure 1

Stem cell grafting improves behavioral performance after chronic chemotherapy. Engraftment of hNSCs improves behavioral performance 1 month after cessation of chronic CYP treatments and transplantation surgery. (A) Schematic of the experimental timeline showing 4 consecutive CYP treatments (100 mg/kg, once weekly) followed by hNSC grafting (week 5), behavioral testing (weeks 9-11) for the novel place recognition (NPR), temporal order (TO), object in place (OiP) and contextual and cued fear conditioning (FC) tasks. After completion of cognitive testing (week 12), animals were euthanized and brains were harvested for the immunohistochemistry and neuron morphology analysis. (B) CYP treatment significantly impaired exploration on a NPR task compared to controls and grafted cohorts (CYP+hNSC) that were not statistically different. (C) CYP treatment impaired preference of prior objects on a TO task compared to controls and grafted cohorts that were not statistically different. (D) Animals subjected to CYP treatment exhibit a trend of altered preference for novel objects at new locations on an OiP task, but were not found to be statistically different compared to other cohorts. (E) One day after baseline and post-training freezing levels were established a context test was administered, where CYP treated cohorts spent significantly decreased percentages of time freezing compared to the control and grafted cohorts (context test bars, E) which were not found to differ. After the initial training phase (48h), the context was changed, which resulted in a substantial reduction in freezing behavior (pre-cue bars, E) that was restored following the tone sound (post-cue test bars, E), indicating intact amygdala function in all groups. All data are presented as Mean ± S.E.M. (N=8 animals each group). *, P=0.01; **, P=0.001; +, P=0.04; ++, P=0.002 compared to CYP group and a, P=0.01 compared to Control group (ANOVA and Bonferroni’s multiple comparisons test).

Figure 2

Survival and differentiation of grafted hNSCs in CYP treated brains. The majority of grafted human cells (HuN+, green) were found ventral to the needle track (Nt) along the corpus callosum (CC) and dorsal to the CA1 subfield within the transplant core (Tc, dotted line, A) with smaller numbers of cell found migrating deeper within the CA1 (A). Higher magnification confocal images (B and C) showing the presence of grafted human cells within the CYP treated brain (Toto3 nuclear counterstain, purple). (D) Differentiation of grafted human cells (HuN+, red) as assessed by dual immunofluorescence staining of phenotypic markers (green). Data indicates the capability of grafted cells to commit to neuronal and astroglial lineages within the microenvironment of the chemotherapy treated brain. Despite relatively larger numbers of grafted cells retaining multipotency (Sox2), immature neurons (DCX), astrocytes (GFAP) and oligo-progenitors (NG2) were found along with their corresponding mature phenotypes (NeuN, S100, Olig2) respectively. Quantification of grafted cell survival by unbiased stereology (E), the yields of individual differentiated neural phenotypes (F) and the percentage and absolute numbers of graft-derived neurons and astroglia (G) are shown. All data are presented as Means ± S.E.M. (N=4 animals per group), scale 50 μM (A), 10 μM (B-C) and 5 μM (D).

Figure 3

Suppression of neuroinflammation following stem cell grafting. Immunohistochemical analysis shows that compared to controls (A), CYP treatment (B) leads to increased numbers of activated microglia (CD68+, red; Toto3 nuclear counterstain, purple) that are reduced in animals receiving stem cells (C). Representative confocal images showing the presence of activated microglia in the hippocampal subfields of the dentate gyrus, (DG), subgranular zone (SGZ) and dentate hilus (DH), with orthogonal reconstructions (inserts) of confocal Z-stacks (A-C). Quantification of activated microglia shows that compared to controls, CYP treatment significantly increased the number of activated microglia in the DH and CA3/CA1 subfields (D). Compared to CYP treated cohorts, animals transplanted with stem cells (CYP+hNSC) were found to have significantly lower numbers of activated microglia in all hippocampal subfields analyzed. Reduced yields of activated microglia in the DH, DG, and CA3/CA1 regions were comparable (or lower) than untreated controls (D). All data are presented as Means ± S.E.M. (N=4 animals per group). *, P=0.01; **, P = 0.001; +, P = 0.001; ++, P = 0.0001 compared to CYP group and a, P=0.03 compared to Control group (ANOVA and Bonferroni’s multiple comparison test). Scale 50 μM (A-C) and 10 μM (inserts).

Figure 4

Transplantation of hNSCs protects granule cell neuronal morphology in the dentate gyrus (DG). Representative images of Golgi-Cox impregnated hippocampal tissue sections from control (A), CYP (B) and CYP+hNSC (C) cohorts reveals the gross disruption to neuronal structure (black) in the DG and CA1 subfields of the hippocampus (nuclear fast red counter-stained) caused by CYP treatment that is resolved in animals receiving stem cells. (D) Representative tracings of granule cell neurons from each cohort superimposed over concentric Sholl circles (20 μM increments). (E) Structural parameters of dendritic morphology (length, volume, complexity) quantified in each cohort showing the CYP-induced reductions in dendritic morphology that were ameliorated by stem cell grafting. Data are presented as Means ± S.E.M. (N=4 animals per group). *, P=0.001; **, P=0.0001; +, P=0.01; ++, P=0.0004 compared to CYP group (ANOVA and Bonferroni’s multiple comparison test). Scale 200 μM (A-C).

Figure 5

Transplantation of hNSCs protects pyramidal neuronal architecture in the CA1. (A) Representative tracings of the basal and apical dendritic tress of CA1 pyramidal cell neurons from each cohort superimposed over concentric Sholl circles (20 μM increments). (B) Structural parameters of basal and apical dendritic morphology (length, volume, complexity) quantified in each cohort showing CYP-induced reductions in dendritic morphology that were ameliorated by stem cell grafting. Data are presented as Means ± S.E.M. (N=4 animals each group). *, P =0.01; **, P = 0.001; ***, P = 0.0001; +, P=0.001; ++, P = 0.0002 compared to CYP group and a, P=0.05 compared to Control group (ANOVA and Bonferroni’s multiple comparison test).

Figure 6

Stem cell grafting preserves dendritic spine density and the number of immature and mature spine morphologies following chronic chemotherapy. Representative images from each cohort showing dendritic spines along Golgi-Cox impregnated granule cell (A) or pyramidal (D) neurons in the DG or CA1 subfields respectively. Quantification of spine morphologies by unbiased stereology shows significantly reduced numbers of both immature (long/thin, mushroom) and mature (stubby) spines in the DG (B) or CA1 (E) of CYP treated animals compared to control and grafted cohorts. The total spine density is reduced significantly in the DG (C) and CA1 (F) of CYP treated animals compared to either control or CYP+hNSC cohorts. All data are presented as Means ± S.E.M. (N=3 animals each group). *, P=0.01; **, P = 0.001; +, P=0.01; ++, P = 0.001 compared to CYP group (ANOVA and Bonferroni’s multiple comparison test). Scale 5 μM (A and D).

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Stem cell transplantation reverses chemotherapy-induced cognitive dysfunction - PubMed (original) (raw)