Post-trauma administration of the pifithrin-α oxygen analog improves histological and functional outcomes after experimental traumatic brain injury

Abstract

Traumatic brain injury (TBI) is a major cause of death and disability worldwide. Programmed death of neuronal cells plays a crucial role in acute and chronic neurodegeneration following TBI. The tumor suppressor protein p53, a transcription factor, has been recognized as an important regulator of apoptotic neuronal death. The p53 inactivator pifithrin-α (PFT-α) has been shown to be neuroprotective against stroke. A previous cellular study indicated that PFT-α oxygen analog (PFT-α (O)) is more stable and active than PFT-α. We aimed to investigate whether inhibition of p53 using PFT-α or PFT-α (O) would be a potential neuroprotective strategy for TBI. To evaluate whether these drugs protect against excitotoxicity in vitro, primary rat cortical cultures were challenged with glutamate (50mM) in the presence or absence of various concentrations of the p53 inhibitors PFT-α or PFT-α (O). Cell viability was estimated by LDH assay. In vivo, adult Sprague Dawley rats were subjected to controlled cortical impact (CCI, with 4m/s velocity, 2mm deformation). Five hours after injury, PFT-α or PFT-α (O) (2mg/kg, i.v.) was administered to animals. Sensory and motor functions were evaluated by behavioral tests at 24h after TBI. The p53-positive neurons were identified by double staining with cell-specific markers. Levels of mRNA encoding for p53-regulated genes (BAX, PUMA, Bcl-2 and p21) were measured by reverse transcription followed by real time-PCR from TBI animals without or with PFT-α/PFT-α (O) treatment. We found that PFT-α(O) (10 μM) enhanced neuronal survival against glutamate-induced cytotoxicity in vitro more effectively than PFT-α (10 μM). In vivo PFT-α (O) treatment enhanced functional recovery and decreased contusion volume at 24h post-injury. Neuroprotection by PFT-α (O) treatment also reduced p53-positive neurons in the cortical contusion region. In addition, p53-regulated PUMA mRNA levels at 8h were significantly reduced by PFT-α (O) administration after TBI. PFT-α (O) treatment also decreased phospho-p53 positive neurons in the cortical contusion region. Our data suggest that PFT-α (O) provided a significant reduction of cortical cell death and protected neurons from glutamate-induced excitotoxicity in vitro, as well as improved neurological functional outcome and reduced brain injury in vivo via anti-apoptotic mechanisms. The inhibition of p53-induced apoptosis by PFT-α (O) provides a useful tool to evaluate reversible apoptotic mechanisms and may develop into a novel therapeutic strategy for TBI.

Keywords: Apoptosis; Controlled cortical impact; PFT-α oxygen analog; Pifithrin-α (PFT-α); Traumatic brain injury (TBI); p53.

Fig. 1. Post-injury administration of PFT-α or PFT-α (O) at 5 h after TBI significantly reduced contusion volume at 24 h

(A) Chemical structures of PFT-α and PFT-α (O). (B) Coronal brain sections (cresyl violet stained) from sham and TBI challenged rats treated with vehicle, PFT-α or PFT-α (O) at 24 h. (C) Contusion volumes induced by TBI at 8 h and 24 h (associated with the primary mechanical injury and secondary apoptotic phases, respectively). All TBI challenged groups were significantly different from the sham group at 8 and 24 h. Whereas the contusion volume of TBI groups were not different from one another at 8 h, by 24 h it was significantly reduced by PFT-α or PFT-α (O) (2mg/kg) treatment compared to the TBI vehicle group. Data are expressed as means ±SEM. * p <0.05, ** p <0.01, ***p<0.001 versus sham group; # p <0.05; ## p <0.01, versus TBI + veh group (n= 5 for each group).

Fig. 2. Post-injury administration of PFT-α (O) at 5 h after TBI significantly decreased FJC positive cells in the cortical contusion region at 24 h

(A) Photomicrographs of FJC-stained regions of interest in sham group, TBI + veh group, TBI + PFT-α and TBI + PFT-α (O) groups. (B) The representative HE-stained coronal section showing the area as indicated by the red square box to compare the fluorescent signals between the 4 groups of rats (the brain section shown is from a sham control). (C) There was a significant decrease in the number of FJC-positive cells in both TBI +PFT- α and TBI +PFT- α (O) groups. The total number of FJC-positive cells was expressed as the mean number per field of view (0.087 mm²). Data are expressed as mean ± SEM. ^### p <0.001 versus TBI+veh group; ⁺⁺⁺ p <0.001 versus TBI+PFT- α group, analyzed by one-way ANOVA. Bar= 50 μm. (n=5 in each group).

Fig. 3. Post-injury administration of PFT-α or PFT-α (O) given at 5h but not 7 h improved functional outcomes as revealed by behavioral evaluation

(A) Motor coordination measured by beam walking test. (B) Neurological function measured by mNSS. (C) Motor asymmetry measured by elevated body swing test (EBST) (D) Sensory-motor function measured by tactile adhesive removal test. Data represent the mean ±SEM. *P <0.05, ***P <0.001 versus sham group; ^#P<0.05, ^###P<0.001 versus TBI +veh group. (n=5 in each group). Note that when PFT was given at 7h post injury, it was much less effective.

Fig. 4. Post-injury administration of PFT-α or PFT-α (O) at 5 h after TBI significantly decreased p53 and Annexin V positive neurons in the cortical contusion region at 8 h

(A) Co-immunohistochemistry of p53 and NeuN in cortical brain tissue. (C) Co-immunohistochemistry of Annexin V and NeuN in cortical brain tissue. p53 or annexin V immunoreactivity is shown in green, and NeuN (a marker for neurons) is shown in red. Yellow labelling indicates colocalization. (B, D) There was a significant decrease in the number of p53 and Annexin V positive neurons in TBI + PFT-α and TBI + PFT-α (O) group, respectively. Data represent the mean ±SEM. **p<0.01; ***p<0.001 versus the sham group; ^##p<0.001; ^###p<0.001 versus the TBI + veh group; ⁺⁺p<0.01; ⁺⁺⁺p<0.001 versus the TBI + PFT-α group. Scale bar=100 μm. (n=5 for each group).

Fig. 5. Expression of p53-regulated mRNAs after CCI injury at 8 h, assessed by RT-PCR

The mRNA levels of PUMA, Bax, Bcl-2, Apaf1, and p21 in brain tissue from sham, vehicle treated, and PFT-α or PFT-α (O) groups were analyzed by RT-PCR. Fold change relative to that of sham animals. PFT-α (O) significantly reduced TBI-induced PUMA mRNA expression in the ipsilateral hemisphere compared with vehicle-treated rats. Data are expressed as means±SEM. ^#p<0.05 vs. TBI + veh group (n= 5 in each group).

Fig. 6. Post-injury administration PFT-α (O) at 5 h after TBI significantly decreased p-p53 and PUMA positive neurons in the cortical contusion region at 8 h

(A) Co-immunohistochemistry of p-p53 and NeuN in cortical brain tissue. (C) Co-immunohistochemistry of PUMA and NeuN in cortical brain tissue. Phospho-p53 or PUMA immunoreactivity is shown in green, and NeuN is shown in red. Yellow labelling indicates colocalization. (B, D) There was a significantly decrease in the number of p-p53 and PUMA positive neurons in TBI + PFT-α (O) group, respectively. Data represent the mean ±SEM. **p<0.01; ***p<0.001 versus the sham group; ^##p<0.001; ^###p<0.001 versus the TBI + veh group; ⁺⁺p<0.01; ⁺⁺⁺p<0.001 versus the TBI + PFT-α group. Scale bar=100 μm. (n=5 for each group).

Fig. 7. PFT-α and PFT-α (O) reduce glutamate-induced excitotoxicity in cortical cultures

(A) Cultured cells were exposed to various concentrations of glutamate (3, 10, 50, 75 and 100 mM) for 24 h. Cell death (%) was measured by LDH release. 50 mM glutamate was chosen as the concentration for testing PFT analogues. (B) PFT- α or PFT- α (O) (3, 10, 20, 30 μM) was added 30 min after cells were exposed glutamate (50 mM) for 24 hrs in primary cultures of neuron/glia. Cell death was measured by LDH activity in culture media which was scaled to the value of maximal death (100%) measured after freeze-thaw treatment of sister cultures. Data are expressed as means±SEM. *p<0.05; **p<0.01; ***p<0.001 compared with the control group; ^#p<0.05; ^##p<0.01; ^###p<0.001 compared with the glutamate treatment (n=3-5 in each group).

Fig. 8. PFT-α or PFT-α(O) reduces glutamate-induced neuronal loss and activation of microglia

(A) Photomicrograph showing nuclear structure revealed by immunocytochemical staining with NeuN and ED1 in control cultures, cultures treated with glutamate (50mM), cultures treated with glutamate (50 mM) and PFT-α (10μM), and cultures treated with glutamate (50 mM) and PFT-α (O) (10μM) at 24 h after glutamate-induced excitotoxicity. Scale bar, 50 μ m. (B, C) Quantitative comparison of NeuN-positive (NeuN⁺) / ED1-positive (ED1⁺) cells in control cultures, glutamate cultures, glutamate cultures treated with PFT-α, and glutamate cultures treated with PFT-α (O). Data are expressed as means±SEM. ***P <0.001 versus control cultures; ###P<0.001 versus glutamate cultures. Bar= 50 μm. (n= 5 in each group).

Fig. 9. PFT-α (O) suppresses glutamate-induced the expression of PUMA in cortical cells

The mRNA levels of PUMA, Bax, Bcl-2, Apaf1and p21 in glutamate-treated (50mM) cortical cultures with PFT-α or PFT-α (O) (10 μM) were analyzed by qRT-PCR. Fold changes relative to that of control cultures are shown. Reduction of the elevated messenger RNA (mRNA) levels of PUMA in primary cultures by PFT-α (O) treatment at 24 hr after glutamate-induced excitotoxicity. PFT-α (O) did not change the level of Bax, Bcl2 Apaf1and p21 genes. Data are expressed as means±SEM. #p<0.05 vs. glutamate group (n= 5 in each group).