Increasing evidence has demonstrated that Ngb plays an important role in neuroprotection against hypoxic/ischemic brain injury, such as stroke and other related neurological disorders [5
]. Our previous studies showed that Ngb overexpression reduces tissue infarction and markers of oxidative stress in a focal stroke mouse model [5
]. Recent reports by others have shown that Ngb gene expression was increased after TBI, but its effects on outcomes were not examined [21
]. In this short study we tested our hypothesis that Ngb over-expression may protect against traumatic brain injury in mice. We performed three sets of experiments to examine (1) changes in brain Ngb protein expression after TBI, (2) the effects of Ngb over-expression in a mechanistic endpoint-oxidative tissue damage, and (3) the neurological outcomes of neurobehavioral deficits and traumatic lesion size at three weeks after TBI. Both Ngb-Tg and WT control mice were examined and compared.
In the first set of experiments, we found that Ngb protein level in the injured brains were significantly increased in both Ngb-Tg and WT mice compared to their sham controls at 6
h after TBI. This examination by immunohistochemistry (Figure A) and western blot analysis (Figure B) was consistent with a report that showed a transient increase of brain Ngb expression with peak at 6
h after TBI in rats. That study also showed Ngb over-expression decreased mechanical injury-induced neuron death [23
]. In our present study, quantification of western blot analysis showed that the basal levels of Ngb protein expression (149% of WT sham control mice) and the CCI-induced increase of Ngb protein levels at 6
h after TBI (196% of WT sham control mice) were significantly higher in Ngb-Tg mouse brains compared to WT TBI controls (147% of WT sham control mice) (Figure1C). While this difference suggested that TBI induces Ngb protein expression, the basal and TBI-induced Ngb protein levels were still significantly higher in Ngb-Tg mouse brains, validating the possible causality between the different brain Ngb protein levels in association with different neurological outcomes after TBI of Ngb-Tg and WT control mice.
Previous studies have demonstrated that Ngb over-expression can reduce hypoxia/ischemia-induced oxidative cell damage in cultured neurons and focal cerebral ischemia in mice [5
]. In the second set of experiments, we tested and compared a common oxidative tissue damage biomarker, 3NT production, in TBI-injured brains of Ngb-Tg and WT control mice. We found that there was a significant reduction of 3NT level at 6
h after TBI in Ngb-Tg mouse brains as compared to WT controls (Figure ), indicating TBI-induced oxidative tissue damage may be diminished by Ngb over-expression.
In the last set of experiments, we examined neurological outcomes of neurobehavioral deficits for up to three weeks after TBI and traumatic brain lesion size at three weeks after TBI. At 0,1, 3, 5, 7, 10, 14, 21
days after TBI, sensorimotor functions were assessed by neurological score and hanging wire tests. Body weight loss was also measured on each test day. Experimental data showed significant deficits in all tests from day 1 to day 7 after TBI. All deficits were recovered close to pre-injury baselines by day 21 after CCI. However, there were no statistically significant differences between Ngb-Tg and WT mice in all assessments during the 3-week TBI recovery period (Table ). We also used Morris Water Maze to assess spatial memory acquisition at 15-21
days after TBI, but we did not find significant differences between Ngb-Tg and WT mice in latency for hidden and visible platform trials (Figure A), as well as in probe trials (Figure ). Lastly, we quantitatively examined traumatic brain lesion volume and found it was significantly reduced in Ngb-Tg mice compared to WT mice at 21
days after TBI (Figure ).
Sensorimotor Function Assessments
Figure 3 Spatial memory acquisitions in WT and Ngb-Tg mice after TBI.A. Morris water maze latencies were measured on days 15-21 for hidden and visible platform trials after TBI. B. Morris water maze latencies were measured on days 15-21 for probe trials after (more ...)
Figure 4 Measurements of cortical lesion volume in WT and Ngb-Tg mice.A. Representative photomicrographs of the traumatic lesions in H&E stained WT and Ngb-Tg mouse brain sections at 21days after TBI. B. Traumatic brain lesion size. Data were (more ...)
In the past two decades neuroprotectants that seek to block or inhibit one specific step in the cascades of TBI have not been very clinically successful [24
]. It has been shown that TBI triggers endogenous protective mechanisms that can prevent or limit brain damage. Methods that seek to augment the brain's own endogenous protection and repair signals may lead to new therapeutic strategies for stroke and related disorders [25
]. Experimental studies from our lab and others have documented that Ngb is one of very few unique molecules for endogenous neuroprotection, which functions in stabilizing neuronal function and prosurvival genes under both normal rest and hypoxic/ischemic conditions, serving to protect against oxidative stress and preserve mitochondrial function [4
]. Furthermore, investigators have recently been attempting to elucidate Ngb gene regulation mechanisms, identifying small molecules that can specifically up-regulate endogenous Ngb protein expression for the development of novel endogenous neuroprotection strategies for treating neurological disorders [19
]. Thus in this study, for the first time, we tested whether Ngb overexpression has neuroprotective effects in TBI model of mice. Results from present work showed: (1) TBI significantly increased Ngb expression in peri-lesion of ipsilateral cortex at 6
h after TBI compared to sham in both WT and Ngb-Tg mice, but the increased Ngb level was significantly higher in Ngb-Tg mice than WT controls; (2) Ngb-Tg significantly reduced the levels of oxidative damage marker 3NT at 6
h after TBI in Ngb-Tg mice compared to WT controls; (3) Ngb-Tg mice exhibited smaller lesion volumes at 3
weeks after TBI compared to WT; (4) however, there were no significant differences in neurobehavioral deficits between the WT and Ngb-Tg mice after TBI for up to three weeks.
Ngb-Tg mice exhibited smaller cortical lesion volumes, suggesting that Ngb overexpression may prevent TBI-induced brain tissue damage. Oxidative stress has a mechanistic role in the pathophysiology of many neurologic diseases, including traumatic brain injury [15
], and previous studies have shown that Ngb over-expression may reduce hypoxia/ischemia-induced oxidative stress in cultured neurons and a focal stroke mouse model [4
]. Additionally, 3NT production at early time points after brain injury is associated with the degrees of TBI-induced oxidative tissue damage [15
]. Thus, we tested and compared 3-NT production levels in the brain tissues of Ngb-Tg mice and WT mice after TBI. The significant reduction of 3-NT production in Ngb-Tg mouse brains suggested that Ngb over-expression may protect TBI brain damage, at least partially, by decreasing oxidative stress, which has been similarly observed in other studies of cells cultures and animal disease models [5
]. Although we did know exactly why there was no effects of Ngb-Tg in oxidative stress reduction in the sham mice, but we may speculate that Ngb may have multiple functions for cell protection by maintaining hemostasis via stress sensing, or transducing signals or direct action. Ngb might respond stress to eliminate excessively elevated oxidative signaling and tissue damage, but is not or less effectively taking action under physiological resting conditions [4
]. Therefore the basal level of 3NT in sham group was not significantly altered as observed in present work.
We acknowledge that the early reduction of 3NT production at 6 hours after TBI in Ngb-Tg mice might be correlated with the lesion reduction at 3
weeks, but other factors likely also contribute to the neuroprotection of Ngb-Tg in TBI. A number of laboratories, including ours, have demonstrated that Ngb is protective against hypoxic/ischemic brain injury. Although the underlying mechanisms remain poorly defined, initial evidence suggests that the neuroprotective effect of Ngb may be linked largely to the structural features related to O2
and NO binding. Furthermore, putative signal transduction and mitochondrial function preservation also may be involved in the protective mechanisms [4
]. However the involved molecular mechanisms need to be investigated in further studies.
It is worthy of noting that, there are two published studies closely related to the present work. A recent study showed that over-expression of Ngb in cultured neurons was neuroprotective against mechanical injury in vitro. This study also showed increased Ngb expression levels with peak time of 6 hours after TBI, and these increased levels correlated to the severity of TBI in rats [23
]. Thus, we picked only the time point of 6 hours after TBI for examining changes of Ngb expression and 3NT production after TBI in present study. One of our previous studies in focal stroke showed oxidative tissue damage biomarker malondialdehyde levels in ischemic hemispheres of Ngb-Tg were significantly reduced compared with wild-type controls at 8 hours and 22 hours after transient focal cerebral ischemia. Brain infarction volumes 1
day and 14
days after stroke were significantly reduced in Ngb-Tg mice. However, there were no significant detectable improvements in sensorimotor deficits for up to 14
days after stroke in Ngb-Tg mice compared with wild-type controls [5
], which was similarly observed in present work in TBI mice.
We are aware that there are a few limitations in this study. First, the total Ngb protein expression levels in our Ngb-Tg mouse line is about 1.5 fold of WT control, which is relatively lower than when the Ngb-Tg mouse line was newly generated 5
years ago [5
]. The decline of the Ngb protein level in Ngb-Tg mice may be mainly due to the endogenous depletion of inserted exogenous DNA fragments or inactivation of its promoter [30
]. However findings from present work are still very informative and suggest that Ngb over-expression might be a new target for TBI therapy strategy development. We are working on a generation of neuron-specific and inducible Ngb-Tg mouse line for future studies of Ngb over-expression effects in TBI neurological outcomes and underlying molecular mechanisms. The second limitation is related to mechanisms. We only detected the reduction of 3-NT production in the Ngb-Tg mouse brains at the early time of 6 hours after TBI, suggesting there might be an antioxidative role for Ngb in vivo. However, for in vivo TBI models, it is difficult to unequivocally prove causality because reduced tissue damage may secondarily contribute to decreased oxidative stress and/or mitochondrial dysfunction. The third limitation is that we failed to detect significant differences in sensorimotor function or spatial memory function recovery between WT and Ngb-Tg mice. In part, this is because the TBI setting in our study might not be severe enough to cause long-term sensorimotor deficits since most of the deficits recovered close to the pre-injury baseline from one to two weeks after TBI. Also, the Ngb protein level in Ngb-Tg mouse brain might not be high enough. Alternatively, we may have simply lacked power and larger numbers of test animals would have helped. Further investigation is warranted to carefully define the role of Ngb in both long-term sensorimotor and cognitive deficits after TBI.