Therapeutic effects of pharmacologically induced hypothermia against traumatic brain injury in mice

Abstract

Preclinical and clinical studies have shown therapeutic potential of mild-to-moderate hypothermia for treatments of stroke and traumatic brain injury (TBI). Physical cooling in humans, however, is usually slow, cumbersome, and necessitates sedation that prevents early application in clinical settings and causes several side effects. Our recent study showed that pharmacologically induced hypothermia (PIH) using a novel neurotensin receptor 1 (NTR1) agonist, HPI-201 (also known as ABS-201), is efficient and effective in inducing therapeutic hypothermia and protecting the brain from ischemic and hemorrhagic stroke in mice. The present investigation tested another second-generation NTR1 agonist, HPI-363, for its hypothermic and protective effect against TBI. Adult male mice were subjected to controlled cortical impact (CCI) (velocity=3 m/sec, depth=1.0 mm, contact time=150 msec) to the exposed cortex. Intraperitoneal administration of HPI-363 (0.3 mg/kg) reduced body temperature by 3-5°C within 30-60 min without triggering a shivering defensive reaction. An additional two injections sustained the hypothermic effect in conscious mice for up to 6 h. This PIH treatment was initiated 15, 60, or 120 min after the onset of TBI, and significantly reduced the contusion volume measured 3 days after TBI. HPI-363 attenuated caspase-3 activation, Bax expression, and TUNEL-positive cells in the pericontusion region. In blood-brain barrier assessments, HPI-363 ameliorated extravasation of Evans blue dye and immunoglobulin G, attenuated the MMP-9 expression, and decreased the number of microglia cells in the post-TBI brain. HPI-363 decreased the mRNA expression of tumor necrosis factor-α and interleukin-1β (IL-1β), but increased IL-6 and IL-10 levels. Compared with TBI control mice, HPI-363 treatments improved sensorimotor functional recovery after TBI. These findings suggest that the second generation NTR-1 agonists, such as HPI-363, are efficient hypothermic-inducing compounds that have a strong potential in the management of TBI.

Keywords: blood–brain barrier; hypothermia; inflammation; neurotensin analogue; sensorimotor function; traumatic brain injury.

FIG. 1.

HPI-363 induced hypothermic effect in normal awake and traumatic brain injury (TBI) mice. The hypothermic effect of NTR1 agonist HPI-363 was assessed in normal C67BL/6 mice without anesthesia and in mice subjected to anesthetic and TBI procedures. Body temperature was measured using a rectal probe, and brain temperature was measured using a brain exclusive probe. (A) Dose-response relationship of HPI-363-induced hypothermic effect in normal mice. Intraperitoneal administration of HPI-363 reduces body core temperature in a dose-dependent manner. Mild to moderate temperature reductions were reached within 30 to 60 min after the drug injection, and the hypothermic effect lasted for various durations depending on the HPI-363 dosage. *p<0.05 vs. saline control; n=3–11/group. (B) HPI-363-induced hypothermic effect in TBI mice. The reduction in body and brain temperature was parallel and consistent. The relatively lower brain temperature was because of the different measurement devices and methods. (C) Using the HomeCageScan system, walking behavior (distance) of sham control mice and hypothermia treated mice were recorded for 3 h. Both HPI-363 and physical cooling treated mice showed reduced locomotion activities. Physical cooling, however, resulted in the most profound effect of reducing walking activity. *p<0.05 vs. normal controls; n=3–5 per group. (D) Shivering behavior in sham control, HPI-363, and physical cooling mice. Shivering was measured during a 3-h period after the treatment in normal animals using the HomeCageScan System. No shivering was triggered by HPI-363-induced hypothermia. *p<0.05 vs. normal controls; n=3–5 per group.

FIG. 2.

HPI-363-induced neuroprotection against traumatic brain injury (TBI). Adult mice were subjected to controlled cortical impact and TBI-induced contusion volume, and cell death was measured 12 h or 3 days after the insult. (A) Nissl stained brain sections of TBI mice at 3 days after TBI. Images show brain sections from a TBI-saline control mouse and a TBI plus HPI-363 treatment mouse (15 min after TBI). The HPI-363 treatment (0.3 mg/kg bolus followed by two supplemental intraperitoneal injections at 0.15 mg/kg, 6-h hypothermia) resulted in a smaller contusion area (*). (B, C) Bar graphs summarize the contusion volume and the loss of hemispheric tissues in the TBI group and the TBI plus HPI-363 group. HPI-363 was administered with 15, 60, 120, or 180 min delay after TBI. The hypothermic treatment with 15 to 120 min delay significantly reduced the contusion volume and the loss of hemispheric tissue. As a control, a group of HPI-363-treated mice was kept in a temperature-controlled incubator to counteract the hypothermic effect. Body temperature in the mice was maintained at 36–37°C during and after TBI. Contusion infarct volumes developed in the mice similar to TBI saline controls. The protective effect disappeared when HPI-363 was administered 180 min after TBI. The contusion volumes and the loss of hemispheric tissues in TBI plus HPI-363 treatment with 15 min delay were still reduced even at 21 days after TBI. *p<0.05 vs. TBI saline group; n=9–10/group. (D) TUNEL staining revealed DNA damage and cell death 12 h after TBI. Total cells were visualized with Hoechst 33342 staining (blue). Massive TUNEL-positive cells (green) were observed in the TBI injured cortical region. HPI-363 treatment resulted in fewer TUNEL-positive cells. (E) The percentage of TUNEL-positive cells among total Hoechst 33342-positive cells in the pericontusion area were counted and summarized in the bar graph. HPI-363 treatment with 15 min to 120 min delay after TBI significantly attenuated cell death. Similar to the contusion volume assay, the hypothermia therapy initiated with a 180 min delay lost the protective effect of TBI-induced cell death. *p<0.05 vs. TBI group; n=9–10/group. Scale bars=20 μM. Color image is available online at www.liebertpub.com/neu

FIG. 3.

HPI-363-induced hypothermia attenuated apoptotic genes in mice after traumatic brain injury (TBI). Expressions of proteins associated with apoptotic cell death were measured using Western blot analysis in the penumbra region 12 h after TBI. (A–D) TBI significantly enhanced expression of the pro-apoptotic genes caspase-3 (A and B) and Bax (C and D). In TBI mice that received HPI-363 (15 min delay), the increases in these pro-apoptotic factors were effectively reduced. (E, F) The expression of bcl-2 in the TBI brain was not significantly altered in the TBI brains although HPI-363 showed a trend of increasing this anti-apoptotic gene. *p<0.05 vs. sham control; #p<0.05 vs. TBI control; n=3 in sham group, n=5 in TBI and TBI plus HPI-363 groups, respectively.

FIG. 4.

HPI-363-induced hypothermia attenuated traumatic brain injury (TBI)-induced blood–brain barrier (BBB) disruption. The functional and morphological integrities of the BBB were measured 12 h after TBI. (A, B) The leakage of Evans blue (EB) was photographed under the TRITC (red) excitation wavelength at 1.25×under a fluorescent microscope. The EB dye positive area (red) was markedly decreased in the HPI-363 treatment group. *p<0.05 vs. TBI group; n=6–7 per group. Scale bars=400 μM. (C, D.) Western blot analysis showed marked immunoglobulin G (IgG) extravasation in the TBI group compared with the sham group. HPI-363 treatment significantly reduced IgG extravasation compared with TBI controls. *p<0.05 vs. sham group; #p<0.05 vs. TBI group; n=3 in sham control, n=5 in TBI group, and n=4 in TBI plus HPI-363 group. (E, F) Immunohistochemical staining of occludin (green), Glut-1 (red), and Hoechst (blue). TBI dramatically reduced occludin expression, indicative of severe BBB damage. HPI-363 treatment showed significant protective effect on occludin expression. *p<0.05 vs. sham group; #p<0.05 vs. TBI group; n=9–10 per group. Scale bars=20 μM. (G, H) Western blot analysis of MMP-9 showed that TBI increased MMP-9 expression while HPI-363 significantly prevented the increase. (I, J) The expression of matrix metalloproteinase (MMP)-2 was not changed in the TBI brain; HPI-363 showed an inhibitory effect on MMP-2 expression. *p<0.05 vs. sham group; #p<0.05 vs. TBI group; n=3–5 per group in G to J assays. Color image is available online at www.liebertpub.com/neu

FIG. 5.

HPI-363-induced hypothermia reduced inflammatory response after traumatic brain injury (TBI). TBI-induced inflammation was evaluated in the pericontusion region 12 h after TBI insult. (A–C) Activated microglia and neuronal cells were detected using immunostaining of Iba1 (green) and NeuN (red) immunofluorescent staining (A). Hoechst 33342 (blue) staining revealed all cells in the region. TBI injury drastically increased Iba1-positive microglia cells while HPI-363 significantly prevented this microglia activation (B). In the TBI brain in the pericontusion region, the number of NeuN-positive neurons was decreased by more than 50%. This neuronal damage was lessened in the HPI-363-treated TBI brain (C). *p<0.05 vs. sham group; #p<0.05 vs. TBI group; n=6–7/group. Scale bars=20 μM. (D) Real-time–polymerase chain reaction (RT-PCR) analysis of inflammatory cytokines. (E–H) Summarized RT-PCR assays. TBI increased proinflammatory cytokines including tumor necrotizing factor (TNF)-α (E) and interleukin (IL)-1β (F), while HPI-363 largely prevented these cytokine increases. Decreased anti-inflammation factor IL-6 was seen in the TBI brain, and HPI-363 maintained the IL-6 mRNA expression near the normal level (G). There was no change in the anti-inflammation cytokine IL-10 after TBI, while HPI-363 treatment substantially enhanced IL-10 mRNA expression (H). *p<0.05 vs. sham group; #p<0.05 vs. TBI group; n=3–5 per group. Color image is available online at www.liebertpub.com/neu

FIG. 6.

HPI-363-induced hypothermia improved sensorimotor functional recovery after traumatic brain injury (TBI). Adhesive dot removal tests and cylinder tests were used to evaluate the sensorimotor functional recovery after TBI. (A, B) The latency to recognize the sticky dot and removal time for the right and left forelimb was recorded at 3 and 7 days after TBI. After TBI damage to the right side of the sensorimotor cortex, both time to feel and time to remove the sticky dot were increased. Mice that received HPI-363 treatment showed significantly improved performance on the removal test even when HPI-363 was administered 2 h after TBI. (C) The cylinder test showed that forelimb activities of mice with TBI were impaired during the first few days after TBI. Mice that received HPI-363 performed significantly better than TBI controls. The difference disappeared 10 days after TBI because of spontaneous recovery of this moderate TBI models. *p<0.05 vs. sham group; #p<0.05 vs. TBI group; n=3–7 per group.

FIG. 7.

HPI-363 treatment improved home cage behavior after traumatic brain injury (TBI). A 24-h continuous monitoring and comprehensive analysis of the behavioral changes of animals in different groups (sham control, TBI control, and TBI plus HPI-363) were performed using the HomeCageSys system (Clever Sys Inc.) Three days after sham and TBI procedures. (A–H) Changes of animal home cage activities quantified from the video recordings. HPI-363 largely corrected many dysfunctional behaviors in animals with TBI. (I) Summary of the percentages of time spent on each behavior during a 12-h monitoring period. *p<0.05 vs. sham group; #p<0.05 vs. TBI group; n=3–6 per group.