Effect of midazolam on the proliferation of neural stem cells isolated from rat hippocampus

Abstract

In many recent studies, the inhibitory transmitter gamma-aminobutyric acid has been shown to modulate the proliferation, differentiation and survival of neural stem cells. Most general anesthetics are partial or allosteric gamma-aminobutyric acid A receptor agonists, suggesting that general anesthetics could alter the behavior of neural stem cells. The neuroprotective efficacy of general anesthetics has been recognized for decades, but their effects on the proliferation of neural stem cells have received little attention. This study investigated the potential effect of midazolam, an extensively used general anesthetic and allosteric gamma-aminobutyric acid A receptor agonist, on the proliferation of neural stem cells in vitro and preliminarily explored the underlying mechanism. The proliferation of neural stem cells was tested using both Cell Counting Kit 8 and bromodeoxyuridine incorporation experiments. Cell distribution analysis was performed to describe changes in the cell cycle distribution in response to midazolam. Calcium imaging was employed to explore the molecular signaling pathways activated by midazolam. Midazolam (30-90 μM) decreased the proliferation of neural stem cells in vitro. Pretreatment with the gamma-aminobutyric acid A receptor antagonist bicuculline or Na-K-2Cl cotransport inhibitor furosemide partially rescued this inhibition. In addition, midazolam triggered a calcium influx into neural stem cells. The suppressive effect of midazolam on the proliferation of neural stem cells can be partly attributed to the activation of gamma-aminobutyric acid A receptor. The calcium influx triggered by midazolam may be a trigger factor leading to further downstream events.

Keywords: gamma-aminobutyric acid A receptor; general anesthetics; hippocampus; midazolam; nerve injury; neural regeneration; neural stem cells; proliferation.

Figure 1

Fluorescence microscopy of nestin immunocytochemistry in cultured neural stem cells (scale bar is 400 μm in A and 200 μm in B). The majority of cells within neurospheres were immunopositive for the neural stem cell-specific cytoskeletal protein nestin (green), whereas only a minority of neurospheres were negative for nestin. The nuclei were also counterstained with 4,6-diamino-2-phenylindole (blue).

Figure 2

Cell Counting Kit 8 cell proliferation test. (A) Dose response studies indicated that mid significantly decreased the proliferation of neural stem cells at a concentration of 30 μM and 90 μM. The repeated administration of 30 μM mid decreased the proliferation of neural stem cells. Pretreatment with 300 μM furo (B) or 10 μM bic (C) partly reversed this repressed proliferation. Data are expressed as mean ± SEM (n = 5 wells/group in A; n = 8 wells/group in B and C). ^aP < 0.05, vs. veh group; ^bP < 0.05, vs. mid group using one-way analysis of variance followed by Newman-Keuls multiple comparison test. mid: Midazolam; veh: vehicle; furo: furosemide; bic: bicuculline.

Figure 3

Bromodeoxyuridine (BrdU) incorporation studies. Rhodamine-conjugated secondary antibody was used to label the cells incorporating BrdU. Mid significantly decreased the percentage of BrdU⁺ neural stem cells at a concentration of 90 μM (A). Repeated administration of 30 μM mid decreased the percentage of cells labeled with BrdU, whereas pretreatment with 300 μM furo (B) or 10 μM bic (C) reversed the repressive effect (original images can be obtained in the supplementary Figure 1 online). Data are expressed as mean ± SEM (n = 6 wells/group). ^aP < 0.05, vs. veh group; ^bP < 0.05, vs. mid group using one-way analysis of variance followed by Newman-Keuls multiple comparison test. mid: Midazolam; veh: vehicle; furo: furosemide; bic: bicuculline.

Figure 4

Cell distribution analysis. Mid (90 μM) decreased the percentage of neural stem cells in S phase, whereas pretreatment with 300 μM furo (A) or 10 μM bic (B) partly or completely reversed this repression. 15 000–20 000 events were measured for every sample. Data are expressed as mean ± SEM (n = 3). ^aP < 0.05, vs. veh group; ^bP < 0.05, vs. mid group using one-way analysis of variance followed by Newman-Keuls multiple comparison test. mid: Midazolam; veh: vehicle; furo: furosemide; bic: bicuculline.

Figure 5

Calcium imaging in neural stem cells. Calcium-dependent fluorescence was elevated immediately upon administration of 90 μM mid and this effect was completely blocked by pretreatment with 10 μM bic. The images were collected at a frequency of 5 seconds per frame. Data are expressed as mean ± SEM (n = 6). ^aP < 0.05, vs. veh group and bic + mid group using two-way analysis of variance. T1: Fluorescence intensity at resting state; T2: fluorescence intensity after administration; mid: midazolam; bic: bicuculline; veh: vehicle.