Restoration of calbindin after fetal hippocampal CA3 cell grafting into the injured hippocampus in a rat model of temporal lobe epilepsy

Abstract

Degeneration of the CA3 pyramidal and dentate hilar neurons in the adult rat hippocampus after an intracerebroventricular kainic acid (KA) administration, a model of temporal lobe epilepsy, leads to permanent loss of the calcium binding protein calbindin in major fractions of dentate granule cells and CA1 pyramidal neurons. We hypothesize that the enduring loss of calbindin in the dentate gyrus and the CA1 subfield after CA3-lesion is due to disruption of the hippocampal circuitry leading to hyperexcitability in these regions; therefore, specific cell grafts that are capable of both reconstructing the disrupted circuitry and suppressing hyperexcitability in the injured hippocampus can restore calbindin. We compared the effects of fetal CA3 or CA1 cell grafting into the injured CA3 region of adult rats at 45 days after KA-induced injury on the hippocampal calbindin. The calbindin immunoreactivity in the dentate granule cells and the CA1 pyramidal neurons of grafted animals was evaluated at 6 months after injury (i.e. at 4.5 months post-grafting). Compared with the intact hippocampus, the calbindin in "lesion-only" hippocampus was dramatically reduced at 6 months post-lesion. However, calbindin expression was restored in the lesioned hippocampus receiving CA3 cell grafts. In contrast, in the lesioned hippocampus receiving CA1 cell grafts, calbindin expression remained less than the intact hippocampus. Thus, specific cell grafting restores the injury-induced loss of calbindin in the adult hippocampus, likely via restitution of the disrupted circuitry. Since loss of calbindin after hippocampal injury is linked to hyperexcitability, re-expression of calbindin in both dentate gyrus and CA1 subfield following CA3 cell grafting may suggest that specific cell grafting is efficacious for ameliorating injury-induced hyperexcitability in the adult hippocampus. However, electrophysiological studies of KA-lesioned hippocampus receiving CA3 cell grafts are required in future to validate this possibility.

FIGURE 1

Schematic of major experiments performed in this study. The experiments mainly include kainic acid (KA) lesions of host animals (illustrated in the center), the dissection and dissociation of fetal hippocampal CA3 and CA1 tissues from donor fetuses into single cell suspensions, grafting of donor cells into the injured CA3 region at 45 days post-KA administration, perfusion of animals, and processing of tissues for Nissl staining and calbindin immunostaining, and measurement of calbindin immunopositive neurons in the dentate granule cell layer and the CA1 pyramidal cell layer using stereology. F, Fimbria; ICV KA, intracerebroventricular kainic acid; S, Subiculum.

FIGURE 2

Extent of hippocampal injury after unilateral intracerebroventricular kainic acid (ICV KA) injection in young adult F344 rats. Figure compares the cytoarchitecture of an intact hippocampus (A1, A2), hippocampus ipsilateral to KA at 45 days post-administration (B1, B2), hippocampus ipsilateral to KA at 6 months post-administration (C1, C2), and hippocampus contralateral to KA at 6 months post-administration (D1, D2). Note the persistent loss of neurons in the CA3 pyramidal cell layer (denoted by asterisks) and the dentate hilus of hippocampi ipsilateral to KA administration at both 45 days and 6 months after KA administration. However, all hippocampal cell layers appear intact in hippocampus contralateral to KA administration. DG, dentate gyrus. Scale bar: A1, B1, C1, D1 = 500 μm; A2, B2, C2, D2 = 200 μm.

FIGURE 3

Calbindin immunoreactivity in the hippocampus of naive rats (A1–A4). Note dense calbindin immunoreactivity in the principal mossy fiber bundle (A2), dentate granule cells (A3), and in the superficial CA1 pyramidal neurons (A4). DG, dentate gyrus; DH, dentate hilus; pMFB, principal mossy fiber bundle; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scale bar: A1 = 500 μm; A2, A3 = 200 μm; A4 = 100 μm.

FIGURE 4

Bilateral loss of hippocampal calbindin immunoreactivity following unilateral intracerebroventricular kainic acid (ICV KA), visualized at 6 months post-administration (A1–B3). A1 is an example of hippocampus ipsilateral to ICV KA demonstrating severe loss of calbindin in dentate granule cells and CA1 pyramidal neurons. Nissl-stained adjacent section however shows intact cell layers in the dentate gyrus and the CA1 subfield (A2). A3–A5 are magnified views from A1, illustrating diminished calbindin immunoreactivity in the principal mossy fiber bundle (pMFB) located in the CA3 region (A3), loss of calbindin in a major fraction of dentate granules (A4), and virtually all CA1 pyramidal neurons (A5) following KA administration. B1 is an example of hippocampus contralateral to ICV KA demonstrating loss of calbindin in dentate granule cells and CA1 pyramidal neurons. B2 and B3 are magnified views of regions from B1 illustrating loss of calbindin immunoreactivity in the dentate gyrus (B2) and CA1 subfield (B3). DG, dentate gyrus; DH, dentate hilus; SO, pMFB, principal mossy fiber bundle; stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scale bar: A1, A2, B1 = 500 μm; A3 = 200 μm; A4, A5, B2, B3 = 100 μm.

FIGURE 5

Calbindin immunoreactivity, and transplant location and morphology in a rat receiving CA3 cell transplants at 45 days post-KA and analyzed at 6 months post-KA. A1 shows the pattern of calbindin immunoreactivity, whereas A2 and A3 demonstrate transplant location and morphology in this rat. A4–A6 are magnified views of regions from A1 demonstrating the robust expression of calbindin in the principal mossy fiber bundle located in the CA3 region (A4), dentate granule cells (A5), and CA1 pyramidal neurons (A6). Similar calbindin immunoreactivity is also seen in the hippocampus contralateral to KA administration and CA3 cell grafting (B1). B2 and B3 are magnified views of regions from B1 demonstrating robust calbindin immunoreactivity in dentate granule cells (B2) and CA1 pyramidal neurons (B3). Note that calbindin expression in this KA treated rat receiving CA3 cell grafts after KA administration is equivalent to calbindin expression observed in an intact control rat (Fig. 3), and much greater than that observed in a rat receiving KA alone (Fig. 4) or a rat receiving KA and CA1 cell grafts (Fig. 6). Asterisks in A2 denote the degenerated CA3 cell layer. DG, dentate gyrus; DH, dentate hilus; ML, molecular layer; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scale bar: A1, A2, B1 = 500 μm; A4 = 200 μm; A3, A5, A6, B2, B3 = 100 μm.

FIGURE 6

Calbindin immunoreactivity, and transplant location and morphology in a rat receiving CA1 cell transplants at 45 days post-KA and analyzed at 6 months post-KA. A1 shows the pattern of calbindin immunoreactivity, whereas A2 and A3 demonstrate transplant location and morphology in this rat. A4–A6 are magnified views of regions from A1 demonstrating the absence of calbindin in the principal mossy fiber bundle located in the CA3 region (A4), expression of calbindin in a fraction of dentate granule cells (A5), and a few CA1 pyramidal neurons (A6). B1 shows the pattern of calbindin immunoreactivity in the hippocampus contralateral to KA administration and CA1 cell grafting (B1). B2 and B3 are magnified views of regions from B1 demonstrating calbindin immunoreactivity in a fraction of dentate granule cells (B2) and CA1 pyramidal neurons (B3). Note that calbindin expression in this rat receiving CA1 cell grafts after KA administration is considerably less than calbindin expression observed in an intact control rat (Fig. 3) and a rat receiving KA and CA3 cell grafts (Fig. 5) but better than calbindin immunoreactivity seen in a rat receiving KA alone (Fig. 4). Asterisks in A1 and A2 denote the degenerated CA3 cell layer. DG, dentate gyrus; DH, dentate hilus; DG, dentate gyrus; ML, molecular layer; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scale bar: A1, A2, B1 = 500 μm; A4 = 200 μm; A3, A5, A6, B2, B3 = 100 μm.

FIGURE 7

Comparison of the total number of calbindin immunoreactive neurons per 0.1 mm³ of tissue in the dentate granule cell layer (A) and the CA1 pyramidal cell layer (B) between hippocampi from intact rats, rats treated with KA alone (lesion only rats), KA treated rats receiving CA3 cell grafts, and KA treated rats receiving CA1 cell grafts. Comparisons utilized one-way analysis of variance (ANOVA) with Student-Newman-Keuls multiple comparisons post hoc test. All data are presented as means ± standard errors (SEM). Note the dramatic loss of calbindin in both dentate granule cells and CA1 pyramidal neurons in rats receiving KA alone and complete restoration of calbindin in KA treated rats receiving CA3 cell grafts. Also, note that CA1 cell grafting in KA treated rats leads to only a partial enhancement of calbindin in the granule cell layer but no increase in CA1 pyramidal cell layer. ***P < 0.001.