Inducible nitric oxide synthase is an endogenous neuroprotectant after traumatic brain injury in rats and mice

Abstract

Nitric oxide (NO) derived from the inducible isoform of NO synthase (iNOS) is an inflammatory product implicated both in secondary damage and in recovery from brain injury. To address the role of iNOS in experimental traumatic brain injury (TBI), we used 2 paradigms in 2 species. In a model of controlled cortical impact (CCI) with secondary hypoxemia, rats were treated with vehicle or with 1 of 2 iNOS inhibitors (aminoguanidine and L-N-iminoethyl-lysine), administered by Alzet pump for 5 days and 1. 5 days after injury, respectively. In a model of CCI, knockout mice lacking the iNOS gene (iNOS(-/-)) were compared with wild-type (iNOS(+/+)) mice. Functional outcome (motor and cognitive) during the first 20 days after injury, and histopathology at 21 days, were assessed in both studies. Treatment of rats with either of the iNOS inhibitors after TBI significantly exacerbated deficits in cognitive performance, as assessed by Morris water maze (MWM) and increased neuron loss in vulnerable regions (CA3 and CA1) of hippocampus. Uninjured iNOS(+/+) and iNOS(-/-) mice performed equally well in both motor and cognitive tasks. However, after TBI, iNOS(-/-) mice showed markedly worse performance in the MWM task than iNOS(+/+) mice. A beneficial role for iNOS in TBI is supported.

Figure 1

iNOS mRNA expression in rats and mice after TBI. iNOS mRNA was detected using RT-PCR. (a) In rats, iNOS mRNA was increased in injured cortex at 2, 6, 24, and 72 hours, and in ipsilateral hippocampus at 2 and 6 hours after TBI plus secondary hypoxemic insult, compared with control (n = 2 animals per group). A 138-bp PCR product is seen. (b) In C57BL/6J mice, iNOS mRNA was increased in injured cortex at 24, 48, and 72 hours after TBI, compared with control (n = 3 animals per group). A 429-bp PCR product is seen. RT-PCR for actin confirmed equal loading of RNA. M, marker; C, uninjured control.

Figure 2

Mean latencies (± SEM) of rats to balance on a beam before and after TBI with secondary hypoxemic insult. Before injury, rats were tested for their ability to balance for up to 60 seconds on a beam. Beginning 1 day after injury, beam-balance latencies were measured daily for 5 consecutive days. All injured groups had shorter latencies (indicating impairment) on day 1 after injury. Shown are saline (filled diamonds), AG (filled circles), and L-NIL (open triangles) treatments. *P < 0.05 vs. sham (open squares; shams were not subjected to CCI or hypoxemia). There were no differences between groups treated with iNOS inhibitors vs. saline after TBI.

Figure 3

Mean latencies (± SEM) of rats to traverse a beam before and after TBI with secondary hypoxemic insult. Before injury, rats were trained to traverse the beam within 5 seconds (Before). Beginning 1 day after injury, latencies were measured daily for 5 days. All injured groups had longer (impaired) latencies on days 1–3 after injury. Shown are saline (filled diamonds), AG (filled circles), and L-NIL (open triangles) treatments. *P < 0.05 vs. sham (open squares; shams were not subjected to CCI or hypoxemia). There were no differences between groups treated with iNOS inhibitors vs. saline after TBI.

Figure 4

The effects of TBI with secondary hypoxemic insult and treatment with iNOS inhibitors on MWM performance in rats. Mean latencies (± SEM) to find a submerged (hidden) platform on days 14–18 after TBI. All injured groups had the longest (most impaired) latencies on day 14 after injury. However, rats treated with either of the iNOS inhibitors (AG or L-NIL) exhibited persistently higher latencies to find the platform on days 16, 17, and 18 after injury, compared with sham (open squares). Shown are saline (filled diamonds), AG (filled circles), and L-NIL (open triangles) treatments. *P < 0.05 vs. sham. Improved performance on visible-platform testing (compared with hidden-platform testing) done on days 19–20 indicates that the deficits seen were not caused by visual impairment.

Figure 5

Number of surviving CA3 hippocampal neurons per HPF (×400) ± SEM at 21 days after TBI with secondary hypoxemic insult in rats treated with saline, AG, or L-NIL, and in shams. The number of surviving CA3 hippocampal neurons was reduced by TBI (*P < 0.05 for saline vs. sham). Treatment with either of the iNOS inhibitors further reduced CA3 hippocampal neuron survival (AG and L-NIL both P < 0.05 vs. *sham or **saline).

Figure 6

Number of surviving CA1 hippocampal neurons per HPF (×400) ± SEM at 21 days after TBI with secondary hypoxemic insult in rats treated with saline, AG, or L-NIL, and in shams. Treatment with either of the iNOS inhibitors reduced CA1 hippocampal neuron survival (*AG and L-NIL both P < 0.05 vs. sham). Rats treated with AG exhibited reduced CA1 neuron survival vs. saline-treated rats after TBI (**P < 0.05).

Figure 7

Assessment of motor function in mice using wire-grip scores (mean ± SEM). Uninjured iNOS^+/+ (open squares) and iNOS^–/– (filled squares) mice performed equally well in this task, as assessed for 5 days. In 2 separate groups of mice, both iNOS^+/+ (open circles) and iNOS^–/– (filled circles) exhibited reduced scores (deficits) in this task after injury (day 1 shows preinjury score; day 2 of testing represents the first postinjury day). *P < 0.05 vs. preinjury scores. There were no differences between iNOS^+/+ and iNOS^–/– groups after injury.

Figure 8

Spatial memory performance using an MWM paradigm in mice. Mean latencies (± SEM) to find a submerged (hidden) platform on days 14–18 after TBI. Uninjured iNOS^+/+ (open squares) and iNOS^–/– (filled squares) mice performed equally well on this task, as assessed for 5 days. After injury, both iNOS^+/+ (open circles) and iNOS^–/– (filled circles) mice showed impaired performance, as assessed for 5 days (vs. either uninjured group). After TBI, iNOS^–/– mice showed markedly impaired performance (*P < 0.05 vs. injured iNOS^+/+ mice on days 15 and 18). Improved performance on visible-platform testing (days 19–20) indicates that the deficits seen were not due to visual impairment.