Role of adult hippocampal neurogenesis in persistent pain

Abstract

The full role of adult hippocampal neurogenesis (AHN) remains to be determined, yet it is implicated in learning and emotional functions, and is disrupted in negative mood disorders. Recent evidence indicates that AHN is decreased in persistent pain consistent with the idea that chronic pain is a major stressor, associated with negative moods and abnormal memories. Yet, the role of AHN in development of persistent pain has remained unexplored. In this study, we test the influence of AHN in postinjury inflammatory and neuropathic persistent pain-like behaviors by manipulating neurogenesis: pharmacologically through intracerebroventricular infusion of the antimitotic AraC; ablation of AHN by x-irradiation; and using transgenic mice with increased or decreased AHN. Downregulating neurogenesis reversibly diminished or blocked persistent pain; oppositely, upregulating neurogenesis led to prolonged persistent pain. Moreover, we could dissociate negative mood from persistent pain. These results suggest that AHN-mediated hippocampal learning mechanisms are involved in the emergence of persistent pain.

Figure 1

Reversible decrease in adult hippocampal neurogenesis by intracerebroventricular administration of antimitotic AraC reversibly blocks tactile and cold allodynia in spared-nerve injury (SNI) neuropathic mice. (A) Diagram of experimental timeline. Group d11 and group d25 were tested for touch, cold (on injured and noninjured paws), and open field at indicated times. Group d11 mice were killed at day 11, and group d25 at day 25 after SNI or sham injury (triangle). Bromodeoxyuridine (Brdu) injections were given for 5 days, 1 week before euthanization (arrow). All animals received lateral ventricle infusion of AraC or saline for 14 days, starting 3 days before peripheral injury. (B) AraC or saline was delivered in the lateral ventricle using an osmotic pump. (C, D)Mean ± SEM, touch and cold sensitivity of injured paw in AraC-SNI (n = 10 up to day 11 and n = 4 for days 14–23), Saline-SNI (n = 8 up to day 11 and n = 4 for days 14–23), and AraC-Sham (n=3 up to day 11 and n=2 for days 14–23)WT mice, during brain perfusion (grey, up to day 11) and after cessation of perfusion (days 14–23). (C) Touch sensitivity (Von Frey [VF] 50% threshold in grams) for the injured paw and during AraC infusion (days 3–11, grey) showed a significant effect of group (F_2,6=37.72, P<0.001), time (F_9,54 = 12.07, P < 0.001), and group-by-time interaction (F_18,54 = 4.23, P < 0.001), as determined by 2-way analysis of variance (ANOVA). T-tests determined that the Saline-SNI animals had significantly lower mechanical thresholds than AraC-SNI or AraC-Sham groups at every time point (days 3–11) (#P<0.001), whereas AraC-SNI and AraC-Sham groups did not differ from each other during AraC infusion. Post hoc 2-way ANOVA on AraC groups only indicate a significant decrease in VF threshold (F_4,68=3.93, P<0.01). After cessation of AraC infusion (days 14–23), AraC-SNI animals begin exhibiting tactile allodynia and in time nearly match that of Saline-SNI. Two-way ANOVA indicated that there was a significant group (F_2,6 = 24.5, P < 0.001), insignificant time (F_3,18 = 2.38, P =0.1), and borderline group-by-time interaction (F_6,18=2.51, P=0.06). AraC-Sham animals significantly differed from both AraC-SNI and Saline-SNI at every time point (days 14–23) (*P < 0.01). Kruskall–Wallis ANOVA by ranks indicated that baseline (BL) thresholds were not different across groups (P = 0.1). (D) Cold sensitivity (acetone test) for the injured paw and during AraC infusion (grey) was significantly higher in Saline-SNI than AraC-SNI or AraC-Sham group at day 5 (U=0.00, Z=3.80, #P<0.001) and day 9 (U=15.00, Z=2.36, #P=0.02). After cessation of AraC, at days 14 and 20, AraC-SNI and Saline-SNI groups were no longer different in cold sensitivity. (E) Examples of labeling for Brdu-cells in the SGZ for each experimental group at time points day 11 and day 25. These images were white balanced in Photoshop. Right insets are examples of double-labeled DCX-Brdu–positive cells. (F) Mean ± SEM, Brdu-labeled cells in different groups (calculated based on 6 slices per animal), at day 11 (during AraC perfusion) and day 25 (after AraC cessation). On day 11, AraC-Sham (n=2) had significantly more Brdu-labeled cells bilaterally in subgranular zone (mean=2632 cells) than AraC-SNI (n=5) (t=4.82, P=0.04, mean=837), whereas on day 25, AraC-Sham had more Brdu cells (mean = 3555) than AraC-SNI (n = 4) (t = 3.36, P = 0.04, mean = 2084). Moreover, on day 11, Saline-SNI (n = 4) mice had significantly greater Brdu-labeled cells as AraC-SNI (n = 5) (t = 4.55, P = 0.04), which was reversed after AraC cessation as illustrated by increased Brdu cells in AraC-SNI at day 25 (n = 4) as compared to AraC-SNI at day 11 (n = 5) (t = 7.68, P < 0.01) (*P < 0.05).

Figure 2

Long-term ablation of adult hippocampal neurogenesis by x-irradiation delays onset of tactile, but not cold, allodynia in spared-nerve injury (SNI) neuropathic mice. (A) Diagram of experimental timeline. Wild-typemice received either x-ray (n=10) or sham (n=10) brain irradiation over 1 week (double arrows; performed at Columbia U., New York city), 96 days before SNI injury. After 12 days of recovery period, all mice were shipped to Chicago (Northwestern U.), quarantined for 84 days, then underwent unilateral SNI injury, and tested for touch, cold (on injured and noninjured paws), open field, and black box emergence at indicated times. All mice were killed 52 days after peripheral injury. Bromodeoxyuridine (Brdu, open circle) injection was given 2 hours before killing (arrow). (B) X-irradiation was directed towards the subgranular zone (SGZ) portion of the hippocampus. (C)Mean ± SEM, touch sensitivity of the SNI-injured paw in Sham and x-ray–radiated mice, monitored for 42 days. There was a significant effect of group (F_1,18=19.71, P<0.0004), time (F_9,162=50.46, P<0.00001), and group-by time interaction (F_9,162=2.58, P<0.009). At every time point, except at baseline (BL) and days 34 and 42, x-ray mice had higher tactile thresholds than sham mice (*P < 0.05). (D) Mean ± SEM, cold sensitivity (acetone test response duration in seconds) of the SNI-injured paw in Sham and x-ray–radiated mice, monitored for 19 days. There was only a significant effect of time (F_6,108 = 10.32, P < 0.00001) but no group or group-by-time interaction effects. (E, F) Doublecortin labeling. Representative slices in Sham-irradiated and SNI-injured mice (E), and x-ray–irradiated and SNI-injured animals (F), in the dorsal (top) and ventral (bottom) dentate gyrus stained for doublecortin. There was almost complete absence of doublecortin-positive cells in the x-ray–irradiated SNI–injured animals. Doublecortin labeling was counted in 2 Sham and 2 x-ray–irradiated mice (10 left and 10 right hemisphere slices per animal). Total doublecortin-labeled cells (left + right) were 1787, 2860 in Sham, and 2, 2 in x-ray–irradiated SNI mice. VF, Von Frey.

Figure 3

Neuropathic and inflammatory persistent pain behaviors are diminished in transgenic mice with decreased adult hippocampal neurogenesis (AHN) (Bone morphogenetic protein [BMP]) (A, B), whereas recovery from inflammatory pain is delayed in transgenic mice with increased AHN (Noggin) (C). (A)Mean ± SEM, touch sensitivity of the spared-nerve injury (SNI) paw in BMP and wild-type (WT) littermates who underwent either SNI or sham surgery (BMP SNI n=8, BMP Shamn=6, WT SNI n=8, WT Shamn=7). Tactile sensitivity of the neuropathic paw was monitored at presurgery baseline (BL) and for subsequent 14 days. BMP-SNI animals did not develop tactile allodynia throughout the period of monitoring. After injury, WT-SNI animals had significantly lower mechanical thresholds from all other groups (group, F_3,25 = 29.17, P < 0.001; time, F_4,100 = 2.43, P = 0.05; group-by-time, F_12,100 = 3.00, P < 0.01) (WT-SNI vs all others post hoc, #P < 0.05). BMP-Sham and WT-Sham animals differed significantly from each other on days 9 through 14 (post hoc, *P<0.05). (B)Mean ± SEM, touch sensitivity of the carrageenan-injected paw in BMP (n = 13) and WT (n = 12) littermates. Tactile sensitivity of the inflamed paw was monitored at preinjury BL and for subsequent 7 days. BMP animals showed significantly less mechanical allodynia in the carrageenan-injected paw compared with WT animals (group effect, F_1,23=20.20 and P<0.001), and this was observed over time (time effect, F_8,176 = 7.85 and P < 0.001, and group-by-time interaction, F_8,176 = 6.66 and P < 0.01) with post hoc differences (*P < 0.05) seen at hour 6 through day 5. (C) Mean ± SEM, touch sensitivity of the carrageenan-injected paw in Noggin (n=5) and WT (n=5) littermates. No significant group differences were observed. However, post hoc comparison shows a small difference at days 4 and 6 (*P < 0.05). VF, Von Frey.

Figure 4

Bone morphogenetic protein (BMP) exhibit heightened depression-like behavior, whereas Noggin shows reduced depression-like behavior, relative to wild-type (WT) littermates. (A) Mean ± SEM, depression-like behavior assessed by time spent immobile during the forced swim test. BMP mice (n = 15) spent significantly more time immobile than WT (n = 15) (t-value₂₈ = 2.73, P < 0.005). In contrast, Noggin mice (n = 15) spent significantly less time immobile than WT (t-value₂₈ = −2.11, P < 0.02). (B) Mean ± SEM, depression-like behavior assessed by the tail suspension test (n = 15 per group, same mice as in A). Neuron-specific enolase (NSE)–BMP animals showed increased tail suspension immobility (t-value₂₈ = 2.10, P = 0.04), and NSE-Noggin animals show reduced tail suspension immobility (t-value₂₈=−2.12, P < 0.02), as compared with WT controls. *P < 0.05. (C) Mean ± SEM, bromodeoxyuridine (Brdu) labeling (n = 5 per group). NSE-BMP animals showed reduced Brdu labeling, and NSE-Noggin animals showed increased Brdu labeling, as compared with WT controls (group F_2,12 = 142.1, P < 0.0001). *P < 0.05. (D) Mean ± SEM, DCX labeling (n = 5 per group). NSE-BMP animals showed reduced DCX labeling, and NSE-Noggin animals showed increase DCX labeling, as compared with WT controls (group F_2,12 = 154.4, P < 0.0001). *P < 0.05. BMP, bone morphogenetic protein.