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Evaluation of Progesterone Receptor Antagonist and Maxi-K Channel Agonist as Neuroprotective in Feeney's Weight Drop Model of TBI
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.355164
Keywords: Anti-progesterone, craniotomy, Maxi-K channel, neuroprotective, progesterone, traumatic brain injury, weight drop apparatus
Neuroprotection in traumatic brain injury (TBI) is an unmet medical need with innumerable failures during translational efforts.[1],[2],[3],[4],[5] The death rate from the TBI is as high as 35%, with most of the admitted persons between 20 and 40 years of age (50%), and even among the TBI survivors, there is a significant impact on the quality of life.[6] Aglepristone is a complete progesterone receptor antagonist used for inducing abortion in bitches.[7] One of the close congeners of this drug, mifepristone, a partial progesterone receptor antagonist, has shown neuroprotective properties in the cerebellar Purkinje cells model.[8] Mifepristone and aglepristone have similar structures with a Tanimoto similarity index of 0.72 [Supplementary Figure S1]. It is unknown if the neuroprotective property of mifepristone would extend to aglepristone too. Aglepristone also has a peculiar feature of binding to progesterone receptor with more affinity than progesterone molecule itself and possibly possesses the neuronal depolarization property of mifepristone.[7],[8] In addition, progesterone's neuroprotective property was inconclusive with variable results in different studies despite showing positive results in preclinical studies.[9],[10],[11] The detrimental effects of inhibiting progesterone's physiological role by administering progesterone inhibitors may be precluded with the requirement to administer neuroprotective drugs for only a short duration in TBI. N-Salicyloyltryptamine (STP) acts through a multi-modal mechanism of action, namely inhibition of voltage-dependent calcium and sodium channels, inhibition of voltage and calcium-dependent potassium channel, enhancement of GABAergic activity, and anti-oxidative property.[12],[13],[14],[15] Interestingly, STP has demonstrated an essential role in activating Maxi-K channels in GH3 cells, which functions as neuronal calcium sensors. Through this, STP possesses a control mechanism for cellular excitability and neurotransmitter release, thereby reducing the neuronal hyperexcitability.[12] The depressant activity seen with this drug can confer a possible advantage in agitated patients of TBI.[16],[17] In TBI's pathogenesis, inflammation plays an important and dominant role in causing secondary injury, and factors such as oxidative stress can aggregate this damage.[18],[19] The STP also has immunomodulatory activity by inhibiting NF-κβ and ERK pathways.[15],[20] Thus, we evaluated the neuroprotective role of aglepristone alone and STP alone in Feeney's weight drop model of TBI.
The study was reported based on ARRIVE 2.0 guidelines.[21] Male Wistar rats (≥6 months of age) weighing 250–280 gm were selected for the study. The reason for selecting male rats was to avoid confounding the protective effective of estrogen found in female rats. The rats were kept one pair per cage at the Advanced Small Animal facility in PGIMER, Chandigarh with a proper provision of hygienic environment, controlled humidity, controlled temperature (23°C ± 2°C), and 12/12 h light/dark cycle. The animals were given access to food and water ad libitum. The approvals were obtained from the Institutional Animal Ethics Committee (81/OAEC/506 (80th), the Institute Biosafety Committee (403/IBC/2015), and Institute Collaboration Committee (No. 79/19-Edu-16/243). The ethical principles laid down by the local regulatory authority were strictly adhered to. All possible efforts were made to minimize the number of animals and invasive procedures to animals. Model induction Right-sided TBI was induced using Feeney's weight-drop model.[22] We developed two instruments namely the head fixing apparatus and weight drop apparatus collaborating with Rolex Instruments, Ambala, India [Supplementary Figure S2]A and [Supplementary Figure S2]B. The description of both of these instruments are provided in the supplementary section. The model development and associated steps of craniotomy with figures have been detailly described in supplementary material and [Supplementary Figure S3]. Craniotomy and weight-drop were performed in all the experimental groups except for the sham in which craniotomy alone was carried out. Adequate watering and warming of the dura mater were undertaken during craniotomy to prevent injury to the brain. Animal grouping Forty-eight animals (Male Wistar rats) were divided into six groups (n = 8 per group). The six groups evaluated in the study were control (tween: 801%), sham (no treatment), aglepristone (10 mg/kg), aglepristone (20 mg/kg), STP (50 mg/kg), and STP (100 mg/kg). The animals were allocated to the treatment groups based on permuted random block randomization. The duration of administering the interventions was 5 days post-induction of the model. The route of administration was intraperitoneal (i.p.). The doses were chosen based on the dose-response characteristics of previous studies.[7],[12],[14],[15] The dose of 10 mg/kg for aglepristone was selected based on the reported dose of aglepristone for abortion in rats.[23] It is interesting to note that the dose of aglepristone used in rats as well as bitches is the same. The pharmacological rationale for 5-day therapy was based on the translational requirement for administering neuroprotective regimens in the clinics. Also, generally, the drug requires at least five administrations to attain a steady-state concentration. The justification of the 21 days follow-up was to evaluate both the acute and sub-acute response of the interventions. The entire study design can be found in [Figure 1].
Chemicals and reagents STP (PubChem CID: 10356316) was synthesized at the Federal University of Sergipe.[16] It was transported to India after obtaining an official import license for the drug import from the Central Drugs Standard Control Organization (CDSCO), Government of India (TL/NZ/17/000290).[24] Aglepristone (PubChem CID: 14153279). All other chemicals and reagents were of analytical grade and were purchased from reputed National and International agencies. The immunohistochemistry kits for BAX (Bcl-2-associated X protein), Bcl2 (B-cell lymphoma 2), and M30 Cytodeath were obtained from Santa Cruz Biotechnology (Catalogue no: SC-526, Lot no: B0107), Thermo Fisher (MS-123-P0, 123P202), and Roche Diagnostics GmbH (12140322001, 11458000), respectively. Assessment time points and outcomes A battery of six neurobehavioral tests, body weight (wt.), histopathological, and immunohistochemistry measurements was undertaken. The neurobehavioral tests were conducted to assess motor strength (grip strength test), motor coordination (rotarod test), comparison between ipsilateral and contralateral motor functioning (cylinder and vibrissae evoked forelimb placement (VEFP) test), overall activity (28-point neurobehavioral test), and spatial memory (Morris water maze test). Detailed methodology used for each neurobehavioral test is provided in the supplementary file. The performance of the animals during the pre training tests can be found in [Supplementary Figure S4] and [Supplementary Figure S5]. All the animals passed the pre-training metrics and were taken forward to the experiment. While deciding the assessment period of the neurobehavioral tests, precautions were taken to avoid more than two assessments in a single day. The assessment timepoints of the neurobehavioral tests were clubbed at week level and were referred to as “End of the week” (EOW) for simplicity. EOW-1, EOW-2, and EOW-3 refer to assessment at the end of the first week, second week, and third week, respectively. Histopathological and immunohistochemistry were carried out at the end of the study [Figure 1]. The body weights were matched at the baseline between the interventions (F (5,42) = 1.742, P = 0.146) [Supplementary Figure S6]. Histopathological and immunohistochemistry assays On the 21st day post-induction of the model, three rats from each group were sacrificed. The area of interest (the parietal lobe, part of the temporal lobe below the parietal lobe, and the adjacent midline area) were dissected from the extracted brain sample. Histopathological (Hematoxylin and Eosin – H and E staining) and immunohistochemistry staining for BAX, Bcl-2, and M30 Cytodeath were performed. [Supplementary material]. The vital organs were assessed at end of study. Statistical analysis The statistical analysis was conducted using R statistical software (version 3.5.2).[25] In addition to R's base package, ggplot2 and reshape2 packages were employed.[26],[27] The Shapiro–Wilk test was used to assess the distribution of normality. No data transformation or outlier deletion was undertaken. The study results were expressed as mean ± SD if data was parametric and as median and interquartile range (IQR) if the data was nonparametric. For comparison of multiple unrelated groups at a time point, one-way ANOVA with the Tukey test's post-hoc test was used for parametric data, and the Kruskal–Wallis test (KWT) with post-hoc Dunn test was used for nonparametric data. P < 0.05 was considered statistically significant for all the comparisons.
The model was successfully induced for all the rats. The steps of craniotomy have been detailed out in the methodology section of the supplementary file. As a part of the model development, we successfully designed two instruments: a head fixing apparatus and a weight drop apparatus. A detailed description of both instruments is present in the supplementary file. Following the model induction, the interventions were administered to the respective groups as planned, and the outcomes were followed up. Assessment of body weight of the rat There were no statistically significant differences in body weight between the interventions at EO1W (F (5,42) = 2.373, P = 0.055), EO2W (F (5,42) = 0.786, P = 0.566), and EO3W (F (5,42) = 1.778, P = 0.138) [Supplementary Figure S6] and [Supplementary Table 1]. The battery of neurobehavioral test For the grip strength test, STP 100 mg/kg showed a significant difference compared to control at EO1W [Table 1] and [Figure 2]. For the rotarod test, aglepristone 10 mg/kg showed a significant difference compared to control, and STP 50 mg/kg showed a worse score and significant difference compared to control at EO1W. For the same test at EO2W, none of the interventions showed significant difference, but at EO3W, all four interventions showed significant difference compared to control. For evaluation of VEFP using the right paw at EO2W and EO3W, all four interventions showed significant difference compared to control. For the cylinder test and VEFP using the left paw, none of the interventions showed a significant different at any timepoint. For 28-point neurobehavioral test at EO1W, none showed a significant difference; at EO2W, aglepristone 10 mg/kg showed a significant difference compared to control. For the same test at EO3W, aglepristone 10 and 20 mg/kg and STP 100 mg/kg showed a significant difference compared to control. For the MWM test evaluated by latency time to reach the platform at E02W, none of the interventions showed a significant difference; at EO3W, aglepristone 10 mg/kg and STP 100 mg/kg showed a significant difference compared to control. For MWM test evaluated by cumulative distance travelled to reach the platform both at EO2W and EO3W, but none of the interventions showed statistical difference.
Histopathological study - H and E and IHC staining Three animals per group were assessed for H and E and IHC staining. All three slides were examined and the findings consistent across the three were reported [Figure 3], [Supplementary Figure 7]. The control group showed loss of granular and molecular layer in H and E staining had positive BAX and M30 Cytodeath staining and occasionally positive Bcl2 staining, thereby demonstrating damage to the neuronal cells and subsequent natural process of regeneration [Figure 3]a. The sham group showed no abnormality in H and E staining, had downregulation of BAX protein, negative M30 Cytodeath and Bcl-2, thereby denoting the absence of neuronal damage [Figure 3]a. In aglepristone 10 mg/kg, H and E staining showed lack of any disruption to the neuronal orientation, BAX downregulation, mild positivity of M30 Cytodeath, and occasional Bcl-2 positivity, thus demonstrating neuronal protection [Figure 3]a. The dose of aglepristone when increased to 20 mg/kg showed positivity of BAX as well as M30 Cytodeath and downregulation of Bcl-2, thereby depicting harm to the neuronal cells [Figure 3]b. STP at 50 mg/kg did not show neuronal protection as depicted by neuronal loss, axonal bulb formation and diffuse axonal damage in H and E staining, mild BAX positivity, M30 Cytodeath positivity, and absence of Bcl2 expression [Figure 3]b. The dose of STP when increased to 100 mg/kg showed neuroprotection as depicted by sign of neuronal recovery, namely haphazard orientation of neurons, mild perivascular lymphocytic infiltration [Supplementary Figure S7], preservation of all the layers other than the mild damage to the molecular layer in H and E staining, downregulation of BAX-2 along with mild positivity M30 Cytodeath staining, and presence of many Bcl-2 staining cells [Figure 3]b.
Assessment of vital organs The H and E staining of lungs, heart, kidney, and liver in all the study groups at the end of the study was according to normal anatomy and representative samples are shown in [Figure 4]a and [Figure 4]b.
TBI remains a mystery to scientists.[28] Time delay following accidents (i.e., accident to incision time) is one of the key factors deciding prognosis following TBI.[29],[30] A neuroprotective agent will essentially help to reduce the death of the neurons following TBI. Many of the neuroprotective drug discovery attempts had previously passed the preclinical stage but failed at the level of late clinical trials.[10],[11],[31],[32],[33] The reason can be multifold.[34],[35],[36],[37] The description of the craniotomy procedure is not very clear in the literature. We have described the exact methodology for performing craniotomy in this paper.[38] The sham did not show any deficit compared to the control, demonstrating the robustness of the surgical procedure [Figure 3]. The control group had a loss of molecular layer and expressed positive BAX and M30 Cytodeath staining, thereby demonstrating neuronal loss [Figure 3].[39] A spectrum of neurobehavioral tests employed in a paced-out manner helps assess the drug's benefit on multiple fronts, thereby avoiding false results.[40] Following TBI, the wt. of the animal decreases, indicating acute stress after injury and eventually the weight increases.[41] Except for sham, for all the intervention groups, the wt. of the rats in the post-injury phase were in decreasing trend. The antagonism of progesterone by aglepristone 10 mg/kg did not cause any detrimental effect in the neurobehavioral and histopathological outcomes of our study, but aglepristone 20 mg/kg did show negative IHC findings. The possible hypothesized mechanism for beneficial effect at 10 mg/kg includes neuronal depolarization, the anti-edematous property, binding and activation of newer sites in the progesterone receptor, and anti-inflammatory effect on the immune cells recruited to the site of action.[7],[8] Downregulation of BAX representing the sign of neuroprotection was found with aglepristone 10 mg/kg. Aglepristone 20 mg/kg is probably harmful, that is, though some positive results were seen with this dose in the neurobehavioral test, a toxic effect also manifests, that is, aglepristone at 20 mg/kg blocks the progesterone more than the threshold level, thereby eliciting the derogatory effects due to its blockage.[42] Doses between 10 and 20 mg/kg need to be explored to arrive at the optimum neuroprotective dose for future studies. STP 50 mg/kg is not beneficial as shown by negative results in the rotarod, histopathological, and IHC tests. STP 100 mg/kg might have modest neuroprotective benefit as seen by positive histopathological and IHC examination and positive results in three neurobehavioral tests, namely grip strength at EO1W; VEFP test at EO2W; MWM test, and rotarod, and 28-point neurobehavioral test at EO3W. The effect is however modest. It importantly lacked beneficial effect for the rotarod test at EO1W, which is considered the best test for assessing the motor deficit following TBI. Moreover, it also failed to demonstrate a dose-response relationship for the dose of 50 mg/kg.[43] Doses between 50 and 100 mg/kg and greater than 100 mg/kg need to be evaluated in further experiments. Similar results of modest efficacy at higher doses and no effect at lower doses were shown by other drugs evaluated in TBI.[44] We had a few limitations in this study. We limited to research to male Wistar rats alone. We did not have a positive control intervention arm. However, considering that there are no effective positive controls available for TBI, this limitation can be justified. We could have kept progesterone as one of the comparator arms, but we did not do it due to the restricted number of animal availability. Genetic expression study with gene encoding for Na+/K+-ATPase α3 subunit for evaluating neuronal depolarization property of aglepristone would have provided additional insights about mechanism but was not undertaken. We did not evaluate the additional dose levels of the drugs. Nevertheless, we had several strengths such as evaluation of drugs acting through a novel mechanism of action in TBI, description of craniotomy procedure in a detailed fashion, employing a battery of neurobehavioral test, detailed follow-up period for outcome, and evaluation of two dose levels for each investigational agent.
The detailed description of craniotomy provided can be utilized in different types of neuroscience experiments. Aglepristone at 10 mg/kg is neuroprotective in an animal model of TBI. STP at 100 mg/kg can have a modest neuroprotective effect, and it is required to evaluate higher doses for further confirmation. Acknowledgments The authors would like to acknowledge the animals involved in the study. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.
[Additional file 1]
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
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