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Transplantation of neural progenitors enhances production of endogenous cells in the impaired brain

Abstract

Grafting of neural progenitors has been shown to reverse a wide variety of neurobehavioral defects. While their role of replacing injured cells and restoring damaged circuitries has been shown, it is widely accepted that this cannot be the only mechanism, as therapy can occur even when an insufficient number of transplanted cells are found. We hypothesized that one major mechanism by which transplanted neural progenitors exert their therapeutic effect is by enhancing endogenous cells production. Consequently, in an allographic model of transplantation, prenatally heroin-exposed genetically heterogeneous (HS) mice were made defective in their hippocampal neurobehavioral function by exposing their mothers to heroin (10 mg kg−1 heroin on gestation days 9–18). Hippocampal damage was confirmed by deficient performance in the Morris maze (P<0.009), and decreased production of endogenous cells in the dentate gyrus by 39% was observed. On postnatal day 35, they received an HS-derived neural progenitors transplant followed by repeated bromodeoxyuridine injections. The transplant returned endogenous cells production to normal levels (P<0.006) and reversed the behavioral defects (P<0.03), despite the fact that only 0.0334% of the transplanted neural progenitors survived and that they differentiated mainly to astrocytes. An immunological study demonstrated the presence of macrophages and T cells as a possible explanation for the paucity of the transplanted cells. This study suggests one mechanism for the therapeutic action of neural progenitors, the enhancement of the production of endogenous cells, pointing to future clinical applications in this direction by use of neural progenitors or by analogous cell-inducing techniques.

Introduction

Neural precursor cell transplantation has been proposed as a mean of cell replacement therapy. Presumably, transplanted neural progenitors migrate to the damaged area, replacing defective cells and restoring damaged circuitries.1, 2 Indeed, grafting of neural progenitors into the hippocampus was shown to reverse various neurobehavioral defects in the impaired animals.3, 4, 5, 6, 7 Recently, it has been also suggested that transplanted neural precursors have trophic properties that induce endogenous regenerative processes in the host brain.8 Obviously, once grafted cells have integrated in the host circuitry, it is practically impossible to differentiate between their effects as cell replacement and their trophic effects.

We hypothesized that one specific mechanism by which neural progenitors exert their trophic therapy on the host brain and behavior is via the induction of endogenous neural precursor production. The mechanism by which neural progenitors might induce such an effect is not known. One possibility is the induction of cytokines by neural progenitors,8, 9, 10 which enhance proliferation of endogenous neural cells.11, 12, 13, 14, 15

Here, we aimed to examine our hypothesis by employing the prenatally heroin-damaged brain model. The heroin model was chosen because, among other effects, it induces deficits that can be related to specific synaptic functions. Accordingly, administration of heroin to pregnant mice induced deficits in hippocampus-related offspring behavior and concomitant cholinergic-related abolishment of protein kinase C (PKC) activation.16, 17, 18, 19, 20, 21

Consequently, in the present study we employed our previously established allographic mouse model for the reversal of neurobehavioral defects by transplantation of neural progenitors toward testing the present question. Consistent with our previous studies, genetically heterogeneous HS/Ibg mice22 were made deficient in the hippocampus-related Morris maze behavior and in its supposed mechanism, PKC function, by exposing them to heroin transplacentally. They were grafted with neural progenitors derived from the same strain to reverse the neurobehavioral deficits. An extensive study was designed to demonstrate the expected low rates of survival and differentiation to neurons, immune evaluation was carried out to ascertain the possible role of rejection and, most importantly, our hypothesis—that the transplanted neural progenitors, before disappearing, induced an increase in endogenous proliferation of cells, restoring damaged circuitries and resulting in neurofunctional recovery—was tested.

Materials and methods

General

Heterogeneous stock (HS/lbg) mice were used in this study since this strain is very prolific, even under drug exposure.23, 24 Four female and one male were housed in each mating cage and maintained under standard conditions of 24° C and a 12-h light–dark cycle. Females were checked daily at 0800. Those that had mated, as evidenced by the existence of a vaginal plug (date was termed as gestational day 1, GD1), were then housed with the other pregnant females. Heroin was administered on GD9–GD18. On GD18, every female was housed individually. The pups were weaned at postnatal day 25 (PN25), segregated by sex and housed in groups of five. On PN35, they were transplanted with neural precursor cells or with Dulbecco's modified Eagle's medium (DMEM) media and divided into three groups for the following three experiments (see summary in Table 1).

Table 1 Experimental design and the purpose of each experiment

Experiment I

Designed to confirm, in the present experimental groups, our previous findings25 on the effects of prenatal heroin exposure and transplantation of neural precursor on cognitive ability. Neural precursors and medium-transplanted mice were tested in the Morris maze on PN90–PN95. A sample group was perfused, and the brains were taken for immunocytochemical assessment of the graft survival and differentiation.

Experiment II

Neural precursors and medium-transplanted mice were stained for T cell and macrophage markers 20 days after transplantation to assess the possible immunological response to the graft.

Experiment III

Beginning 2 h after transplantation, and continuing for the following 4 days, mice received two daily injections of the mitotic marker bromodeoxyuridine (BrdU), for a total of 10 injections, a dose which has been shown to be a valid marker of neural progenitors in the brain and which avoids nonproliferating cells labeling artifacts.26 Four hours after the last injection, the mice were perfused and brains were taken for immunocytochemical evaluation of the newly born cells in the dentate gyrus.

Specific methods

Prenatal heroin administration

Heroin administration paradigm was based on our previous studies showing neurobehavioral deficits after its prenatal administration.17, 23 Pregnant female mice received a daily subcutaneous injection of 10 mg kg−1 of heroin or saline on GD9–GD18, the period when most brain structures develop.24 The drug was not given after that period in order to prevent neonatal withdrawal, which would adversely affect offspring–maternal interaction.27

Isolation of neural precursors and growth of neurospheres

This procedure is based on the studies of Ben-Hur et al.28, 29 For the derivation of neural progenitors, newborn (PN1–PN2) HS/Ibg mice were used. Cerebral cortices were minced, digested in 0.025% trypsin for 20 min and dissociated with a 5 ml pipette into a single-cell suspension. Debris was removed by centrifuging after addition of 4% bovine serum albumin. Then the pallet was suspended in serum-free F12/DMEM medium supplemented with 10 mg% human apo-transferrin, 1 mM sodium-pyruvate, 0.05% bovine serum albumin, 10 ng ml−1 D-biotin, 30 nM sodium selenite, 20 nM progesterone, 60 μM putrescine, 25 μg ml−1 bovine insulin, 2 mM L-glutamine and 25 μg ml−1 gentamycin (all from Sigma, St Louis, MO, USA). The viability of the cells was monitored throughout the incubation using Trypan blue. The cells were plated 10 × 106 cells/T-75 uncoated flask and incubated in a CO2 air jacket incubator for 6–8 days until grafting. The cells were supplemented daily with 10 ng ml−1 of basic fibroblast growth factor 2 (R&D, Minneapolis, MN, USA) and 20 ng ml−1 of epidermal growth factor 2 (R&D). Under these conditions, most cells died and approximately 0.2% of cells proliferated into clusters of small round cells that grew into floating spheres. After 3 days of incubation, the cells were centrifuged at 800 r.p.m. for 8 min and were resuspended in half of the original volume and incubated for 3 more days. As already described, these spheres consisted mainly of PSA-NCAM+, nestin+ and NG2(−) cells that generated GFAP+ astrocytes, GalC+ oligodendrocytes and few neurofilament+ neurons upon differentiation.28, 29

Sphere transplantation

On PN35, the mice treated prenatally with heroin/saline where transplanted with mouse neurospheres. Animals were anesthetized with an intraperitoneal injection of 80 mg kg−1 pentobarbital and grafted on a stereotaxic apparatus with 2500 neurospheres (100–200 cells per neurosphere) at a volume of 2–5 μl using a 10 μl Hamilton syringe into each hippocampus at the following coordinates: 1.8 mm posterior to bregma, ±1.5 mm lateral from the midline and 1.7 mm below calvarium.17, 30 DMEM media was chosen as the control solution. The issue of an appropriate control has been coped with since the days of grafting of embryonic differentiated cells, as opposed to neural progenitors. Transplantation of dead cells or tissues destined to be metabolized in the brain appeared to have an averse effect on the control group. Nevertheless, such a control may be called for in a future extensive experiment with several control groups so that all variables may be controlled for.

Morris water maze test

Morris maze testing began on PN90 and continued for 5 days. Both the apparatus and the test procedure that were originally developed for rats31 were adapted to enable testing with mice, as previously described.32 The test was performed in a tank with a diameter of 87 cm filled with opaque white water (by adding powdered milk) at 24° C. A clear platform, 10 × 8 × 9 cm high, was submerged 1 cm under water. In the place test, the mice were given two blocks of four trials on each day of the test. In all trials, the mouse was given 60 s to swim, find the platform and climb onto it. The mouse stayed on the platform for 20 s until placed in the water for another trial. Mouse that failed to find the platform in 60 s was manually placed on the platform until the next trial. The time to reach the platform (latency) was recorded. On the fifth day, the mice were given only one block of four trials of the place test followed by one block of four trials of the spatial probe test. In the latter test, the platform was removed and the extinction of the learned behavior was evaluated as the sequential decline in the proportion of the distance swum in the quadrant of the missing platform, over the four 60-s trials. To control for the possible effect of swimming ability on the results, the swimming speed during each of the trials was calculated.

Immunocytochemical evaluation of the transplanted cells in the host brain

A sample group of four animals, which were behaviorally tested, were immunocytochemically evaluated to determine the fate of the transplanted cells. In this group, 25 μg ml−1 BrdU was added to the growth media 72 and 48 h prior to transplantation, so that the transplanted cells could be identified in the host brain. After behavioral testing, the mice were anesthetized with an overdose of pentobarbital and perfused transcardially with 4% paraformaldehyde (Gadot, Netanya, Israel). The brains were then removed and postfixed overnight at 4° C in 4% paraformaldehyde, cryoprotected in 30% sucrose for another 24 h, and finally deep frozen in liquid nitrogen and stored in −70° C. The whole hippocampal area was cut into coronal 10 μm frozen sections. Every sixth section was mounted. Sections were fixed in acetone for 10 min in −20° C, dried, and incubated in 0.3% H2O2 in methanol for 15 min. After phosphate-buffered saline washes, the sections were incubated with 0.1 mg ml−1 proteinase K for 15 min. The sections were treated with 4 N HCl for 10 min and washed to neutralize the pH. Sections were incubated with anti-BrdU (clone Bu20a, 1:20 dilution; Dako, Glostrup, Denmark) overnight at 4° C. A goat anti-mouse immunoglobulin G secondary antibody, conjugated to Alexa 488 (Molecular Probes-Invitrogen, Carlsbad, CA, USA; 1:100), was added for 50 min at room temperature. Counterstaining was carried out with 4,6-diamidino-2-phenylindole. For double labeling, rabbit anti-GFAP (Dako, 1:100) or rabbit anti-neurofilament (Chemicon, Temecula, CA, USA; 1:100) was added and detected by Cy5-conjugated goat anti-rabbit antibody (Jackson, West Grove, PA, USA; 1:100). Mouse anti-neuronal nuclei was also used for double labeling (Chemicon; 1:200) together with a rat anti-BrdU antibody (Axyll, Westbury, NY, USA; 1:200) and detected with fluorescein isothiocyanate-conjugated goat anti-rat antibody and Cy5-conjugated goat anti-mouse antibody (both from Jackson; 1:100).

Coronal sections (10 μm) were cut spanning the entire rostral/caudal width of the hippocampus. Every sixth section was stained for BrdU and counted. The total number of BrdU+ cells was extrapolated for the entire hippocampus. For immunofluorescent analysis, BrdU-positive cells were first examined with fluorescent microscopy (Olympus, Melville, NY, USA) for double labeling with neurofilament or GFAP. Confirmation of double labeling was performed on a confocal microscope (Leica, Wetzlard, Germany).

Detection of macrophages and T cells in the host hippocampus

Twenty days after transplantation, control and neural-precursor-transplanted mice were anesthetized and perfused as described above. Coronal sections (10 μm) from the hippocampal area were further fixed in 4% paraformaldehyde for 20 min. After washing the sections in phosphate-buffered saline, they were blocked in 10% normal goat serum for 1 h at room temperature and incubated with rat anti CD-3 or rat anti-MAC-2 antibodies (1:100; Serotec, Oxford, UK) overnight at 4° C. A goat anti-rat immunoglobulin G secondary antibody, conjugated to Alexa 488 (Molecular Probes-Invitrogen; 1:100), was added for 50 min at room temperature. Counterstaining was performed with 4,6-diamidino-2-phenylindole.

BrdU administration and identification of newly born cells in the dentate gyrus

The third group of mice, which were transplanted with neural precursors on PN35, received two daily intraperitoneal injections of 50 mg kg−1 BrdU (Amersham, Buckinghamshire, UK) beginning on the day of grafting and continuing throughout the next 4 days. The commonly applied dose of BrdU (50 mg kg−1 in one daily injection for up to 12 days) has shown no physiological side effects such as weight loss or behavioral changes.26 In our case, 60 mg kg−1 day−1 was administered as two daily injections of 30 mg kg−1 for 5 days. Four hours after the last injection, the mice were perfused and the brains sectioned and stained with an anti-BrdU antibody, similar to the procedure described above. BrdU-positive cells were counted through the rostral/caudal extent of the dentate gyrus (granular cell layer+hilus). All BrdU-positive cells were quantified as described above for the immunocytochemical evaluation of the transplanted cells.

Statistical analysis

Data are presented as means and s.e., with differences between treatments established by multivariate analysis of variances, followed by Tukey test for post hoc comparisons between groups where appropriate. It should be noted that similar significance levels were shown with a two-tailed t-test for all major findings. Sex differences were considered in the analysis and in agreement with earlier work with prenatal heroin treatment,16, 17 the initial analysis of variance did not identify any significant interaction between treatment and sex; accordingly results from males and females are presented together. Significance for all tests was assumed at the level P<0.05.

Results

Experiment I

Morris water maze performance

In line with our previous work, mice exposed prenatally to heroin displayed impaired Morris water maze performance, requiring more time than controls to reach the platform (Figure 1a; P<0.009); the gap between the control and the treated offspring remained unchanged throughout the entire test period (no interaction of treatment × day). Groups of mice were transplanted with neural precursor cell spheres into the hippocampus bilaterally. Neural sphere transplantation in normal mice had no effect on Morris water maze performance. However, in mice that were exposed prenatally to heroin, the neural progenitors totally reversed the deficits evoked by prenatal heroin exposure. Differences in Morris maze performance did not reflect alterations in swimming ability, as we did not find any differences in the total swimming speed when the platform was removed for the spatial probe test (Table 2). We also did not detect significant differences among groups for extinction in the spatial probe test (Figure 1b): across all groups, there was a progressive reduction in the time spent in swimming in the quadrant in which the platform had been located (P<0.008), but the trial-to-trial variability precluded finding significant intergroup differences in the extinction rate.

Figure 1
figure1

Effects of prenatal heroin exposure and subsequent neural progenitors transplantation on performance in the Morris water maze. Data for latency to find the platform (a) represent mean and s.e. compiled from eight trials on days 1–4 and four trials on day 5 (see Materials and methods). P<0.009 for the difference between heroin+sham vs control+sham and control+NP. Heroin+NP did not differ from control+sham and control+NP. In (b), the platform was removed and the proportion of swimming distance in the quadrant that had contained the platform was determined; values represent mean and s.e. compiled across four trials conducted on day 5. P<0.008 for the global differences between trials (contributed by trial 4—post hoc test). There were no significant differences between groups in this test. Heroin+NP, offspring who were exposed prenatally to heroin and grafted with neural precursors (n=20); control+NP, offspring who were exposed prenatally to saline and grafted with neural precursors (n=13); heroin+sham, offspring who were exposed prenatally to heroin and grafted with media (n=22); control+sham, offspring who were exposed prenatally to saline and grafted with media (n=13).

Table 2 Swimming speed

The transplant survival rate and fate

At 45 days post-transplantation, upon completion of behavioral testing, the mice were killed for histopathological assessment of grafted cells. Identification of grafted cells was enabled by labeling them prior to transplantation with BrdU. Quantification of surviving BrdU-labeled transplanted cells showed a mean number of 334±49 cells per brain. Considering that each animal was grafted with approximately 106 cells (5000 neurospheres divided between the two hippocampi, each containing 200 cells), this indicates a 0.0334% survival rate of the initial amount of transplanted cells. These were mostly localized within the hippocampal formation (Figure 2a), although a few cells could still be found along the corpus callosum and needle tract. A further study of these surviving cells revealed that 21.6% (72±0.5) had differentiated to astrocytes, as shown by GFAP labeling (Figure 2b), whereas no neuronal marker neurofilament or neuronal nuclei positive grafted could be found indicating a total absence of graft-derived neurons in the host dentate gyrus.

Figure 2
figure2

Identification of the transplanted cells in the host brain. (a) BrdU-labeled cells light color in the host hippocampus. (b) Transplanted BrdU-labeled cell expressing the GFAP astrocytes marker (dark). Bars: (a) 50 μm; (b) 10 μm. Confocal microscopy was used in (b). Fluorescent microscopy was used for all other micrographs. BrdU, bromodeoxyuridine.

Experiment II

Macrophages and T cells in the transplanted hippocampus

To examine whether the loss of transplanted cells was due to graft rejection, immunofluorescent stains were performed for CD-3T cells and for MAC-2 macrophages to identify the immune response toward the graft. Twenty days after transplantation of neural progenitors, all animals showed a very eminent presence of CD-3-positive (Figures 3a and b) and MAC-2-positive (a galactose-specific lectin—Figures 3c and d) cells around the hippocampal area (especially the CA1 area), the corpus callosum and the needle tract, indicating the presence of T-cell phenotypes33 and inflammatory and mature murine macrophages,34 respectively. No immune cells were observed in the sham-transplanted control groups (Figures 3e and f).

Figure 3
figure3

Immune cells in the neural-progenitors-transplanted mice. Identification of CD-3+ (a) and MAC-2+ (c) corpus callosal cells of neural-progenitors-transplanted mice, shown with 4,6-diamidino-2-phenylindole nuclear counterstain. (b and d) Low power field images of (a) and (c) respectively. (e and f) Low power field images of the sham-transplanted control, stained with CD-3+ and MAC-2+, respectively, showing no immune cells. CC, corpus callosum; HI, hippocampus. Bars: (a–f) 50 μm.

Experiment III

Enhancement of endogenous cell proliferation in the dentate gyrus via neural progenitors transplantation to heroin-impaired offspring

The performance in the Morris water maze memory task is related to the rate of neurogenesis in the dentate gyrus of the hippocampus. Therefore, we counted the number of host brain proliferating neural progenitors in the dentate gyrus, as indicated by uptake of BrdU by the cells.

In heroin-exposed offspring there were 31% less BrdU-positive cells in the dentate gyrus as compared to control animals (370.5 cells mean heroin+sham, as opposed to 537.43 cells mean control). However, when transplanted with neural progenitors, the number of BrdU-positive cells in the dentate gyrus of heroin-exposed offspring returned to normal (79% increase as compared to sham-grafted heroin-exposed) and even showed some, albeit not statistically significant, overshoot compared to control levels (P<0.02, for the prenatal exposure × transplantation, interaction, analysis of variance). Control animals (not exposed to heroin prenatally) grafted with media or with neural progenitors did not differ in the number of BrdU-positive cells in the dentate gyrus and consequently their results here are pooled. Representative immunocytochemical pictures are shown in Figure 4. The quantitative differences between the neural progenitors and sham-grafted mice can be seen in Figure 5.

Figure 4
figure4

Neurogenesis in the dentate gyrus in transplanted versus control heroin-exposed mice. The incorporation of BrdU into host neural precursors in the dentate gyrus was examined. Few BrdU-labeled cells were observed (light stain, Alexa 488) in the granular cell layer of mice exposed prenatally to heroin and sham operated (a), but significantly more in mice grafted with neural progenitors (b). Bars: (a and b) 10 μm. BrdU, bromodeoxyuridine.

Figure 5
figure5

Quantification of endogenous cells in neural-progenitors-transplanted mice. Number of newly born cells (BrdU-labeled) in prenatal heroin-exposed mice grafted with neural progenitors (686.67±84) as compare to those injected with heroin and media only (370.5±40) and control mice (537.43±66). P<0.05 for the reduction of heroin+sham from control levels. *P<0.006 for the difference of heroin+NP from heroin+sham levels. Two-way analysis of variance. Control (pooled), offspring exposed prenatally to saline and grafted with media (n=4) or neural progenitors (n=3); heroin+sham, offspring exposed prenatally to heroin and grafted with media (n=8); heroin+NP, offspring exposed prenatally to heroin and grafted with neural progenitors (n=9). BrdU, bromodeoxyuridine.

Discussion

The present study attempts to resolve the apparent paradox of the successful reversal of neurobehavioral defects by neural progenitors transplantation on the one hand and, and on the other, the host brain paucity, primarily in xenographic and allographic studies, of visible transplanted cells, particularly neurons; a paucity, which according to the present findings, may be related to an immune reaction. We suggest that the production of new cells in the dentate gyrus is reduced by the insult (prenatal heroin exposure in the present study), and that the transplanted neural progenitors restore production of endogenous neural cells.

Heroin exposure during prenatal development was chosen as the insult that produces the neurobehavioral birth defects for the present model but, as was shown in models of similar teratogens, it does not produce gross morphological aberrations.35, 36 Although heroin is a major ‘hard’ drug of abuse in the US as well as in many other countries,37, 38 it was not chosen for its significance as a teratogen but rather for the fact that prenatal heroin has been shown, in our model, to produce specific defects in the mouse septohippocampal cholinergic innervation-related behaviors and the specific mechanism of this defect has been identified and extensively described. In particular, heroin-induced deficits in eight arm and Morris maze behaviors.16, 17, 18, 19, 20, 21, 39, 40, 41 Both of these behavioral tests are affected by the cholinergic septohippocampal projection.42, 43, 44 On the biochemical/molecular level, prenatal heroin induced both pre- and postsynaptic hyperactivity in the hippocampal cholinergic innervation,16, 17, 23, 45 converging on a total abolishment of the specific cholinergic-induced activation of PKCγ.16, 21, 41, 46 This appears to be a major mechanism of these behavioral defects.

The reversal of the PKCγ inactivation and behavioral deficits with neural progenitors was recently demonstrated41 and the behavioral deficits and their reversal were confirmed in the present study. Although PKC analysis could not be replicated as the brains were needed for immunocytochemical evaluations, based on previous studies,41 the behavioral results suggest that PKC inactivation occurred and was reversed by neural progenitors transplantation. Here we took the heroin model a step further, revealing another mechanism by which this drug exerts its teratogenic effect—inhibiting endogenous production of cells in the dentate gyrus. A central enigma in stem cell research is the possibility of a successful restoration of normalcy in the face of lower than expected visible transplanted cells in the host brain. Although this phenomenon was mostly shown in allographic and xenographic transplantation and/or grafting including our own,41, 47, 48 it was reproduced even with autographic neural progenitors.3 It was shown not only in a Parkinson's model, where a small number of surviving cells are expected and sufficient to produce reversal,47 but even in other models such as hippocampal neurofunctional deficits.48 Thus, to enable survival in allographic and xenographic models, immunosuppression became a routine procedure.9

In the present study we were able to demonstrate clearly this effect by employing an allographic model of neural progenitor transplantation to enhance rejection. HS/Ibg is a HS mouse stock made by crosses of eight inbred strains22 and was since kept heterozygous via a maximum outbreeding program, creating a strain very suitable for allographic transplantation. As already shown in a previous study,41 the survival rate of the grafted neural precursor cells was low and the prelabeled-transplanted cells differentiated mainly into glia. Because morphological defects are expected to be small, we could preclude any transplant-derived neuromorphological regenerative element in the beneficial effect of the graft.

In keeping with the literature,49 this scarcity of transplanted cells is expected to owe itself to immune rejection. MAC-2 (a galactose-specific lectin) is characteristically expressed by inflammatory and mature murine macrophages,34 and CD-3 indicates a presence of T-cell phenotype.33 These cells are the key mediators of graft rejection50 and their presence in the neural progenitors-transplanted brains coupled with their total absence sham-transplanted brains provides evidence of rejection processes resulting from neural progenitors transplantation.

Importantly, despite this rejection, reversal took place on the behavioral and cellular levels, confirming our hypothesis that one important mechanism by which transplanted neural progenitors exert their therapeutic effect is via the enhancement of endogenous cells production, as supported by a related study on bone marrow culture.51 To further substantiate this hypothesis, it should be noted that studies have shown that inflammatory processes have a negative effect on cell proliferation.52, 53 As such, molecular and behavioral recovery can be stimulated despite the fact that, as a result of the inflammatory and rejection processes, many of the transplanted cells are eventually absorbed by the host immune system. Further study ascertaining the fate of the endogenous cells produced as pertaining to the neurobehavioral recovery is still needed. Since in the present study the endogenous cells were only studied soon after their genesis, their fate obviously cannot yet be known. It can only be noted that they are positive for markers like Ki67 (a marker of the cell cycle), doublecortin (a marker for migrating neuronal cells) and nestin (marker of proliferating and migrating cells), all of which are downregulated as the cells start to differentiate.

It is important to note that even if a large proportion of the endogenous cells induced differentiated top glia and not neurons, it will still bear great therapeutic significance.

Whereas the importance of endogenous differentiation to neurons is obvious, there is a growing awareness of the role of glial cells, mainly astrocytes, in the reversal of neurobehavioral deficits.4, 6 Astrocytes are located close to the neural stem cells in the adult dentate gyrus54 and can express several factors that are able to independently increase proliferation.55, 56 Of particular relevance to the present study is the clear connection found between astrocytes, learning and PKCγ.57 Thus, this suggests the mechanism for behavioral reversal, given the well-known connection between PKCγ and Morris maze performance.58, 59, 60, 61, 62, 63 Thus, neural progenitors-induced endogenous differentiation can contribute to the therapeutic effect, even if that differentiation is not neuronal.

The mechanisms by which prenatal heroin exposure causes reduction in the production of endogenous cells are not yet known, though previous studies provide some indications. Muscarinic manipulation implicated the muscarinic signaling cascade, involving G proteins, Ca2+ elevation and PKC, in the proliferation of neural precursors.64 In our model, prenatal heroin exposure induced defects of the cholinergic innervation,16, 17, 23, 45 terminating in PKC inactivation,16, 21, 41, 46 thus serving as a possible explanation for prenatal heroin inhibition of endogenous neural precursors proliferation. It is possible that the muscarinic signaling cascade may be part of, and/or play a synergistic role to, the cytokine system, whose role in neurogenesis has been suggested by other investigators.11, 12, 13, 14, 15 Whether neurogenesis was causative of biochemical recovery, most importantly of PKC, or merely parallel thereof, remains a subject of future investigation.

In conclusion, allographic transplantation of neural progenitors was shown to restore neurobehavioral defects induced by an insult, in the present case, prenatal heroin exposure, despite lower than expected demonstrable transplanted neural progenitors-originating cells in the host brain. To explain this paradox, we have shown that a possible central mechanism for the therapeutic action of the neural progenitors is the induction, directly or indirectly, of endogenous cells production in neurogenic regions, originally depleted by the insult. Further insight can be gained in the future through very extensive studies where proliferation of endogenous cells, as well as the extent of grafted cells, immune markers and cytokines, is evaluated at more temporal points, including at the time of expression of behavioral correction. Additionally, the fate of the endogenous cells thus produced and their therapeutic mechanisms, whether it is by rescuing existing cells (through differentiation to glia) or by replacing lost cells and restoring damaged circuitries (through differentiation to neurons), remains the subject of future investigations.

References

  1. 1

    Snyder EY, Yoon C, Flax JD, Macklis JD . Multipotent neural precursors can differentiate toward replacement of neurons undergoing targeted apoptotic degeneration in adult mouse neocortex. Proc Natl Acad Sci USA 1997; 94: 11663–11668.

    CAS  Article  Google Scholar 

  2. 2

    Bjorklund A, Lindvall O . Cell replacement therapies for central nervous system disorders. Nat Neurosci 2000; 3: 537–544.

    CAS  Article  Google Scholar 

  3. 3

    Toda H, Takahashi J, Iwakami N, Kimura T, Hoki S, Mozumi-Kitamura K et al. Grafting neural stem cells improved the impaired spatial recognition in ischemic rats. Neurosci Lett 2001; 316: 9–12.

    CAS  Article  Google Scholar 

  4. 4

    Shear DA, Tate MC, Archer DR, Hoffman SW, Hulce VD, Laplaca MC et al. Neural progenitor cell transplants promote long-term functional recovery after traumatic brain injury. Brain Res 2004; 1026: 11–22.

    CAS  Article  Google Scholar 

  5. 5

    Qu T, Brannen CL, Kim HM, Sugaya K . Human neural stem cells improve cognitive function of aged brain. NeuroReport 2001; 12: 1127–1132.

    CAS  Article  Google Scholar 

  6. 6

    Zhang H, Lu A, Zhao H, Li K, Song S, Yan J et al. Elevation of NMDAR after transplantation of neural stem cells. NeuroReport 2004; 15: 1739–1743.

    CAS  Article  Google Scholar 

  7. 7

    Yanai J, Ben-Hur T, Slotkin TA, Katz S . Neural progenitors for the reversal of heroin neurobehavioral teratogenicity. Proceedings of the 3rd Annual Meeting of the International Society for Stem Cell Research, San Francisco, CA, USA, 2005, p 259.

  8. 8

    Ourednik J, Ourednik V, Lynch WP, Schachner M, Snyder EY . Neural stem cells display an inherent mechanism for rescuing dysfunctional neurons. Nat Biotechnol 2002; 20: 1103–1110.

    CAS  Article  Google Scholar 

  9. 9

    Yan J, Welsh AM, Bora SH, Snyder EY, Koliatsos VE . Differentiation and tropic/trophic effects of exogenous neural precursors in the adult spinal cord. J Comp Neurol 2004; 480: 101–114.

    Article  Google Scholar 

  10. 10

    Lu P, Jones LL, Snyder EY, Tuszynski MH . Neural stem cells constitutively secrete neurotrophic factors and promote extensive host axonal growth after spinal cord injury. Exp Neurol 2003; 181: 115–129.

    CAS  Article  Google Scholar 

  11. 11

    Gage FH . Mammalian neural stem cells. Science 2000; 287: 1433–1438.

    CAS  Article  Google Scholar 

  12. 12

    Fallon J, Reid S, Kinyamu R, Opole I, Opole R, Baratta J et al. In vivo induction of massive proliferation, directed migration, and differentiation of neural cells in the adult mammalian brain. Proc Natl Acad Sci USA 2000; 97: 14686–14691.

    CAS  Article  Google Scholar 

  13. 13

    Battista D, Ferrari CC, Gage FH, Pitossi FJ . Neurogenic niche modulation by activated microglia: transforming growth factor beta increases neurogenesis in the adult dentate gyrus. Eur J Neurosci 2006; 23: 83–93.

    Article  Google Scholar 

  14. 14

    Aberg MA, Aberg ND, Hedbacker H, Oscarsson J, Eriksson PS . Peripheral infusion of IGF-I selectively induces neurogenesis in the adult rat hippocampus. J Neurosci 2000; 20: 2896–2903.

    CAS  Article  Google Scholar 

  15. 15

    Emsley JG, Hagg T . Endogenous and exogenous ciliary neurotrophic factor enhances forebrain neurogenesis in adult mice. Exp Neurol 2003; 183: 298–310.

    CAS  Article  Google Scholar 

  16. 16

    Steingart RA, Abu-Roumi M, Newman ME, Silverman WF, Slotkin TA, Yanai J . Neurobehavioral damage to cholinergic systems caused by prenatal exposure to heroin or phenobarbital: cellular mechanisms and the reversal of deficits by neural grafts. Brain Res Dev Brain Res 2000; 122: 125–133.

    CAS  Article  Google Scholar 

  17. 17

    Steingart RA, Silverman WF, Barron S, Slotkin TA, Awad Y, Yanai J . Neural grafting reverses prenatal drug-induced alterations in hippocampal PKC and related behavioral deficits. Brain Res Dev Brain Res 2000; 125: 9–19.

    CAS  Article  Google Scholar 

  18. 18

    Izrael M, Van der Zee EA, Slotkin TA, Yanai J . Cholinergic synaptic signaling mechanisms underlying behavioral teratogenicity: effects of nicotine, chlorpyrifos, and heroin converge on protein kinase C translocation in the intermedial part of the hyperstriatum ventrale and on imprinting behavior in an avian model. J Neurosci Res 2004; 78: 499–507.

    CAS  Article  Google Scholar 

  19. 19

    Yanai J, Avraham Y, Levy S, Maslaton J, Pick CG, Rogel-Fuchs Y et al. Alterations in septohippocampal cholinergic innervations and related behaviors after early exposure to heroin and phencyclidine. Brain Res Dev Brain Res 1992; 69: 207–214.

    CAS  Article  Google Scholar 

  20. 20

    Yanai J, Pick CG, Rogel-Fuchs Y, Zahalka EA . Alterations in hippocampal cholinergic receptors and hippocampal behaviors after early exposure to nicotine. Brain Res Bull 1992; 29: 363–368.

    CAS  Article  Google Scholar 

  21. 21

    Yaniv SP, Naor Z, Yanai J . Prenatal heroin exposure alters cholinergic receptor stimulated translocation and basal levels of the PKCbetaII and PKCgamma isoforms. Brain Res Bull 2004; 63: 339–349.

    CAS  Article  Google Scholar 

  22. 22

    McClearn GE, Wilson JR, Meredith W . The use of isogenic and heterogenic mouse stock in behavioral research. In: Lindzey G, Thiessen DD (eds). Contributions to Behavior-Genetic Analysis: The Mouse as a Prototype. Appleton-Century-Crofts: New York, 1970, pp 3–22.

    Google Scholar 

  23. 23

    Steingart RA, Barg J, Maslaton J, Nesher M, Yanai J . Pre- and postsynaptic alterations in the septohippocampal cholinergic innervations after prenatal exposure to drugs. Brain Res Bull 1998; 46: 203–209.

    CAS  Article  Google Scholar 

  24. 24

    Rodier PM . Critical periods for behavioral anomalies in mice. Environ Health Perspect 1976; 18: 79–83.

    CAS  Article  Google Scholar 

  25. 25

    Yanai J, Ben-Hur T, Katz S, Ben-Shaanan T . Heroin neurobehavioral teratogenicity in mice is reversed by neural progenitor grafts. Proceedings of the Satellite Meeting of The International Society for Neurochemistry/European Society for Neurochemistry, Isola de San Servolo, Venice, Italy, 2005.

    Google Scholar 

  26. 26

    Cooper-Kuhn CM, Kuhn HG . Is it all DNA repair? Methodological considerations for detecting neurogenesis in the adult brain. Brain Res Dev Brain Res 2002; 134: 13–21.

    CAS  Article  Google Scholar 

  27. 27

    Riley EP, Barron S . The behavioral and neuroanatomical effects of prenatal alcohol exposure in animals. Ann N Y Acad Sci 1989; 562: 173–177.

    CAS  Article  Google Scholar 

  28. 28

    Einstein O, Karussis D, Grigoriadis N, Mizrachi-Kol R, Reinhartz E, Abramsky O, et al. Intraventricular transplantation of neural precursor cell spheres attenuates acute experimental allergic encephalomyelitis. Mol Cell Neurosci 2003; 24: 1074–1082.

    CAS  Article  Google Scholar 

  29. 29

    Ben-Hur T, Einstein O, Mizrachi-Kol R, Ben-Menachem O, Reinhartz E, Karussis D et al. Transplanted multipotential neural precursor cells migrate into the inflamed white matter in response to experimental autoimmune encephalomyelitis. Glia 2003; 41: 73–80.

    Article  Google Scholar 

  30. 30

    Yanai J, Pick CG . Neuron transplantation reverses phenobarbital-induced behavioral birth defects in mice. Int J Dev Neurosci 1988; 6: 409–416.

    CAS  Article  Google Scholar 

  31. 31

    Morris R . Developments of a water-maze procedure for studying spatial learning in the rat. J Neurosci Methods 1984; 11: 47–60.

    CAS  Article  Google Scholar 

  32. 32

    Rogel-Fuchs Y, Newman ME, Trombka D, Zahalka EA, Yanai J . Hippocampal cholinergic alterations and related behavioral deficits after early exposure to phenobarbital. Brain Res Bull 1992; 29: 1–6.

    CAS  Article  Google Scholar 

  33. 33

    Steward M, Bishop R, Piggott NH, Milton ID, Angus B, Horne CH . Production and characterization of a new monoclonal antibody effective in recognizing the CD3 T-cell associated antigen in formalin-fixed embedded tissue. Histopathology 1997; 30: 16–22.

    CAS  Article  Google Scholar 

  34. 34

    Reichert F, Rotshenker S . Deficient activation of microglia during optic nerve degeneration. J Neuroimmunol 1996; 70: 153–161.

    CAS  Article  Google Scholar 

  35. 35

    Yanai J . Comparison of early barbiturate and ethanol effects on the CNS. Subst Alcohol Actions Misuse 1981; 2: 79–91.

    CAS  PubMed  Google Scholar 

  36. 36

    Yanai J, Rosselli-Austin L, Tabakoff B . Neuronal deficits in mice following prenatal exposure to phenobarbital. Exp Neurol 1979; 64: 237–244.

    CAS  Article  Google Scholar 

  37. 37

    National Institute on Drug Abuse DoEaSARTI. National Household Survey on Drug Abuse: Main Findings 1992. National Institute on Drug Abuse Division of Epidemiology and Prevention Research US Department of Health and Human Services Public Health Service Alcohol Drug Abuse and Mental Health Administration: Rockville, MD, 1994.

  38. 38

    Baranea Z, Taichman M, Rahav G . Drug and alcohol abuse in Israel-Epidemiological study PORY—Public Opinion Studies in Israel. 1990.

  39. 39

    Levin ED, Addy N, Baruah A, Elias A, Christopher NC, Seidler FJ, et al. Prenatal chlorpyrifos exposure in rats causes persistent behavioral alterations. Neurotoxicol Teratol 2002; 24: 733–741.

    CAS  Article  Google Scholar 

  40. 40

    Levin ED, Addy N, Nakajima A, Christopher NC, Seidler FJ, Slotkin TA . Persistent behavioral consequences of neonatal chlorpyrifos exposure in rats. Brain Res Dev Brain Res 2001; 130: 83–89.

    CAS  Article  Google Scholar 

  41. 41

    Katz S, Ben-Hur T, Ben-Shaanan TL, Yanai J . Reversal of heroin neurobehavioral teratogenicity by grafting of neural progenitors. J Neurochem (in press).

  42. 42

    Parent MB, Baxter MG . Septohippocampal acetylcholine: involved in but not necessary for learning and memory? Learn Mem 2004; 11: 9–20.

    Article  Google Scholar 

  43. 43

    Toumane A, Durkin T, Marighetto A, Galey D, Jaffard R . Differential hippocampal and cortical cholinergic activation during the acquisition, retention, reversal and extinction of a spatial discrimination in an 8-arm radial maze by mice. Behav Brain Res 1988; 30: 225–234.

    CAS  Article  Google Scholar 

  44. 44

    McNamara RK, Skelton RW . The neuropharmacological and neurochemical basis of place learning in the Morris water maze. Brain Res Brain Res Rev 1993; 18: 33–49.

    CAS  Article  Google Scholar 

  45. 45

    Abu-Roumi M, Newman ME, Yanai J . Inositol phosphate formation in mice prenatally exposed to drugs: relation to muscarinic receptors and postreceptor effects. Brain Res Bull 1996; 40: 183–186.

    CAS  Article  Google Scholar 

  46. 46

    Beer A, Slotkin TA, Seidler FJ, Yanai J . Nicotine therapy in adulthood reverses the synaptic and behavioral deficits elicited by prenatal exposure to phenobarbital. Neuropsychopharmacology 2005; 30: 156–165.

    CAS  Article  Google Scholar 

  47. 47

    Studer L, Tabar V, McKay RD . Transplantation of expanded mesencephalic precursors leads to recovery in parkinsonian rats. Nat Neurosci 1998; 1: 290–295.

    CAS  Article  Google Scholar 

  48. 48

    Jeltsch H, Yee J, Aloy E, Marques Pereira P, Schimchowitsch S, Grandbarbe L et al. Transplantation of neurospheres after granule cell lesions in rats: cognitive improvements despite no long-term immunodetection of grafted cells. Behav Brain Res 2003; 143: 177–191.

    Article  Google Scholar 

  49. 49

    Barker RA, Widner H . Immune problems in central nervous system cell therapy. NeuroRx 2004; 1: 472–481.

    Article  Google Scholar 

  50. 50

    Brevig T, Holgersson J, Widner H . Xenotransplantation for CNS repair: immunological barriers and strategies to overcome them. Trends Neurosci 2000; 23: 337–344.

    CAS  Article  Google Scholar 

  51. 51

    Munoz JR, Stoutenger BR, Robinson AP, Spees JL, Prockop DJ . Human stem/progenitor cells from bone marrow promote neurogenesis of endogenous neural stem cells in the hippocampus of mice. Proc Natl Acad Sci USA 2005; 102: 18171–18176.

    CAS  Article  Google Scholar 

  52. 52

    Monje ML, Toda H, Palmer TD . Inflammatory blockade restores adult hippocampal neurogenesis. Science 2003; 302: 1760–1765.

    CAS  Article  Google Scholar 

  53. 53

    Zhu F, Qian C . Berberine chloride can ameliorate the spatial memory impairment and increase the expression of interleukin-1beta and inducible nitric oxide synthase in the rat model of Alzheimer's disease. BMC Neurosci 2006; 7: 78.

    Article  Google Scholar 

  54. 54

    Song H, Stevens CF, Gage FH . Astroglia induce neurogenesis from adult neural stem cells. Nature 2002; 417: 39–44.

    CAS  Article  Google Scholar 

  55. 55

    Rudge JS, Alderson RF, Pasnikowski E, McClain J, Ip NY, Lindsay RM . Expression of ciliary neurotrophic factor and the neurotrophins-nerve growth factor, brain-derived neurotrophic factor and neurotrophin 3-in cultured rat hippocampal astrocytes. Eur J Neurosci 1992; 4: 459–471.

    Article  Google Scholar 

  56. 56

    Nakayama T, Momoki-Soga T, Inoue N . Astrocyte-derived factors instruct differentiation of embryonic stem cells into neurons. Neurosci Res 2003; 46: 241–249.

    CAS  Article  Google Scholar 

  57. 57

    Van der Zee EA, Luiten PG, Disterhoft JF . Learning-induced alterations in hippocampal PKC-immunoreactivity: a review and hypothesis of its functional significance. Prog Neuropsycopharmacol Biol Psychiatry 1997; 21: 531–572.

    CAS  Article  Google Scholar 

  58. 58

    Colombo PJ, Gallagher M . Individual differences in spatial memory among aged rats are related to hippocampal PKCgamma immunoreactivity. Hippocampus 2002; 12: 285–289.

    Article  Google Scholar 

  59. 59

    Colombo PJ, Wetsel WC, Gallagher M . Spatial memory is related to hippocampal subcellular concentrations of calcium-dependent protein kinase C isoforms in young and aged rats. Proc Natl Acad Sci USA 1997; 94: 14195–14199.

    CAS  Article  Google Scholar 

  60. 60

    Douma BR, Van der Zee EA, Luiten PG . Translocation of protein kinase Cgamma occurs during the early phase of acquisition of food rewarded spatial learning. Behav Neurosci 1998; 112: 496–501.

    CAS  Article  Google Scholar 

  61. 61

    Van der Zee EA, Compaan JC, Bohus B, Luiten PGM . Alterations in the immunoreactivity of muscarinic acetylcholine receptors and colocalized PKC gamma in mouse hippocampus induced by spatial discrimination learning. Hippocampus 1995; 5: 349–362.

    CAS  Article  Google Scholar 

  62. 62

    Van der Zee EA, Compaan JC, de Boer M, Luiten PGM . Changes in PKC gamma immunoreactivity in mouse hippocampus induced by spatial discrimination learning. J Neurosci 1992; 12: 4808–4815.

    CAS  Article  Google Scholar 

  63. 63

    Van der Zee EA, Strosberg AD, Bojus B, Luiten PGM . Colocalization of muscarinic acetylcholine receptors and protein kinase C gamma in rat parietal cortex. Mol Brain Res 1993; 28: 152–162.

    Article  Google Scholar 

  64. 64

    Ma W, Maric D, Li BS, Hu Q, Andreadis JD, Grant GM et al. Acetylcholine stimulates cortical precursor cell proliferation in vitro via muscarinic receptor activation and MAP kinase phosphorylation. Eur J Neurosci 2000; 12: 1227–1240.

    CAS  Article  Google Scholar 

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Acknowledgements

This work was supported by USPHS Grant ES13147, the United States-Israel Binational Science Foundation BSF2005003 and the Israeli Anti-Drug Authority.

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Correspondence to J Yanai.

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Ben-Shaanan, T., Ben-Hur, T. & Yanai, J. Transplantation of neural progenitors enhances production of endogenous cells in the impaired brain. Mol Psychiatry 13, 222–231 (2008). https://doi.org/10.1038/sj.mp.4002084

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Keywords

  • endogenous cells
  • heroin
  • hippocampus
  • immunological rejection
  • mice
  • neural progenitors therapy

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