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. Author manuscript; available in PMC: 2022 Oct 1.
Published in final edited form as: Exp Neurol. 2021 Jun 25;344:113792. doi: 10.1016/j.expneurol.2021.113792

Agonism of the α7-Acetylcholine Receptor/PI3K/Akt Pathway Promotes Neuronal Survival after Subarachnoid Hemorrhage in Mice

Ari Dienel 1, Remya A Veettil 1, Kanako Matsumura 1, H Alex Choi 1,2, Peeyush Kumar T 1, Andrey S Tsvetkov 2,3,4, Jaroslaw Aronowski 2, Pramod Dash 5, Spiros L Blackburn 1, Devin W McBride 1,*
PMCID: PMC8359014  NIHMSID: NIHMS1723379  PMID: 34181928

Abstract

Subarachnoid hemorrhage (SAH) results in severe neuronal dysfunction and degeneration. Since the nicotinic acetylcholine α7 receptors (α7-AChR) are involved in neuronal function and survival, we investigated if stimulation of α7-AChR would promote neuronal survival and improve behavioral outcome following SAH in mice. Male mice subjected to SAH were treated with either galantamine (α7-AChR agonist) or vehicle. Neurobehavioral testing was performed 24 hours after SAH, and mice were euthanized for analysis of neuronal cell death or a cell survival (PI3K/Akt) signaling pathway. Neuron cell cultures were subjected to hemoglobin toxicity to assess the direct effects of α7-AChR agonism independent of other cells. Treatment with the α7-AChR agonist promoted neuronal survival and improved functional outcomes 24 hours post-SAH. The improved outcomes corresponded with increased PI3K/Akt activity. Antagonism of α7-AChR or PI3K effectively reversed galantamine’s beneficial effects. Tissue from α7-AChR knockout mice confirmed α7-AChR’s role in neuronal survival after SAH. Data from the neuronal cell culture experiment supported a direct effect of α7-AChR agonism in promoting cell survival. Our findings indicate that α7-AChR is a therapeutic target following SAH which can promote neuronal survival, thereby improving neurobehavioral outcome. Thus, the clinically relevant α7-AChR agonist, galantamine, might be a potential candidate for human use to improve outcome after SAH.

Keywords: subarachnoid hemorrhage, neuron survival, galantamine, nicotinic acetylcholine receptor

Introduction

Nearly thirty thousand Americans experience aneurysmal subarachnoid hemorrhage (aSAH) each year and the mortality rate is up to 67% (Zacharia et al., 2010). Furthermore, the socio-economic burden is large since the average age of individuals affected by aSAH is 45–55 years (de Rooij et al., 2007) and about 30% of survivors develop neurological and cognitive impairments (Connolly et al., 2012; Cossu et al., 2014; Foreman, 2016).

Experimental studies have shown that SAH causes severe neuronal death (Croci et al., 2021; Fujii et al., 2013; Hasegawa et al., 2011; Shi et al., 2017) which may lead to the clinical manifestation of delayed infarction and delayed neurological deficits (Macdonald, 2014). Animal models of SAH have indicated that improving neuronal survival leads to improved functional outcome (Croci et al., 2021; Shi et al., 2017), and so promoting neuronal survival may provide a clinical benefit.

Agonists of nicotinic acetylcholine α7 receptors (α7-AChR) have been shown to be a therapeutic target for brain hemorrhages, including traumatic brain injury (Gatson et al., 2015), intracerebral hemorrhage (Katsuki and Matsumoto, 2018), and SAH (Duris et al., 2011). For example, administration of acetylcholinesterase inhibitors that increase synaptic acetylcholine levels and activate cholinergic receptors have been reported to be beneficial in experimental traumatic brain injury (Chen et al., 1996; Yu et al., 2015). Thus, α7-AChR stimulation may be a robust therapeutic target to reduce the pathologies resulting from aSAH. Furthermore, agonism of α7-AChR can promote neuronal cell survival after traumatic brain injury (Gatson et al., 2015) and intracerebral hemorrhage (Krafft et al., 2012). Based on these and other findings, we hypothesized that α7-AChR agonism will promote neuronal survival and improve neurobehavioral outcomes after SAH in mice. Additionally, we hypothesized that the PI3K/AKT pathway is crucial for the mechanism of α7-AChR agonism, and as such, interference with this pathway would reverse the beneficial effects of α7-AChR agonism.

Materials and Methods

All animal experiments were approved by the local Animal Welfare Committee, conducted in compliance with the National Institutes of Health Guidelines for the Use of Animals in Neuroscience Research, and are reported following the Animal Research: Reporting in Vivo Experiments guidelines.

Sixty-two adult male C57BL/6J mice (28–36 g) and 6 P0 C57BL/6J pups were used. Only male mice were used since a recent study showed that α7-AChR agonism is beneficial to male and female mice after SAH (Dienel et al., 2021). Mice were housed in a humidity- and temperature-controlled room with a 12 hour light-dark cycle. Mice were given ad libitum access to food and water. Mice were electronically randomized into groups prior to surgery. Sample sizes were estimated with SigmaPlot 11.0 using data from previous publications, α=0.05, and β=0.2. Individuals performing functional assessment, measurement of outcomes, or data analysis were kept blinded to the experimental groups.

To examine the effects of α7-AChR agonism on behavior and neuronal survival, mice (n=8/group) were allocated into sham, SAH+Vehicle (normale saline), SAH+Galantamine (α7-AChR agonist, Tocris Bioscience), or SAH+Galantamine+MLA (methyllycaconitine citrate, α7-AChR antagonist, Tocris Bioscience). MLA is 40-fold more selective for α7-AChR than its other cholinergic receptors (α4β2 or α6β2). Neurobehavioral performance (n=8/group) was assessed one day after SAH prior to euthanasia and tissue collection for histology (n=6/group).

To test the effect of α7-AChR knockout on neuronal degeneration, we utilized brain tissue from α7-AChR knockout mice (subjected to either sham, SAH+Vehicle, or SAH+Galantamine in a previous study (Dienel et al., 2021) and stained for fluoro-jade C.

To study the mechanism by which galantamine reduces neuronal degeneration and improves neuronal survival, mice were allocated into either sham, SAH+Vehicle (5% mannitol), SAH+Galantamine, SAH+Galantamine+MLA, or SAH+Galantamine+BAY80-6946 (PI3K inhibitor, Selleckchem). Mice (n=6/group) were used to examine functional performance and protein expressions of the PI3K/AKT/CC3 pathway. BAY80-6946 was chosen as the antagonist for PI3K since it is 1000-fold more selective for PI3K than mTOR (FDA New Drug Application 209936). Neurobehavioral performance was assessed one day after SAH prior to euthanasia and tissue collection for Western blot.

To test the specific effect of galantamine on neuronal cells, we investigated neuronal degeneration and apoptosis using primary neuron cultures. Briefly, cortical tissues from P0 mouse pups were dissected, dissociated, and plated on tissue-culture plates (6×105/well for immunocytochemistry) coated with poly-D-lysine (A-003-E, BD Biosciences), as we have previously done (Moruno-Manchon et al., 2020). Primary cortical neurons were grown in Neurobasal Medium (A3582901, Thermo Fisher Scientific) supplemented with B-27 (A3582801, Thermo Fisher Scientific), GlutaMAX (35050061, Thermo Fisher Scientific), and penicillin-streptomycin (15070063, Thermo Fisher Scientific). Cultures were grown for 15 days before being subjected to experimental conditions. Wells received MLA (1.3 μM) or saline (10 μL), then 30 minutes later were given galantamine (10 μM) or saline (0.8 μL), and then 1 hour later hemoglobin (3 μM) (Chen-Roetling and Regan, 2016) or saline (10 μL) administered. Twenty-four hours after hemoglobin administration, cells were prepared for either western blot (media removed, lysis buffer added, and then cells collected) or immunocytochemistry (fixed with 4% paraformaldehyde) (Moruno-Manchon et al., 2020).

Subarachnoid Hemorrhage Model

The endovascular perforation model of subarachnoid hemorrhage was performed as previously described (Dienel et al., 2020). Perforation was confirmed by respiratory distress. Vehicle, galantamine (0.5 mg/kg), MLA (0.1 mg/kg), and BAY80-6946 (0.1 mg/kg, 5% mannitol) were intranasally administered 1 and 8 hours post-SAH. Briefly, 5 μL was given, alternating each nostril, for a total of 30 μL with a one minute delay between administrations. Animals not recovering from surgery were excluded and replaced. Animals which recovered from surgery but did not survive until neurobehavioral testing were excluded from all analysis. After euthanasia and brain removal, the circle of Willis was observed for blood and used to confirm SAH.

Neurobehavioral Performance

All animals underwent functional performance evaluation using a 24-point neuroscore, beam walking, T-maze, and rotarod one day post-surgery. The neuroscore test evaluates sensorimotor function (Matsumura et al., 2019). Beam walking examines the mouse’s ability to traverse a round rod (McBride et al., 2015). T-maze assesses working memory; mice were given 10 individual trials to walk to the end of a “T” and choose to turn into either the left or right arm. The choices for each trial were recorded and the number of spontaneous alterations (i.e. choosing to explore a different arm from the previous trial) are reported. Rotarod tests the mouse’s motor skills. Briefly, mice were trained using two trials of a constant 5 RPM. Mice were then tested using a 5 RPM starting speed with an 8 RPM acceleration and then a 5 RPM starting speed with a 16 RPM acceleration (2–3 trials each). Each trial was a total of 2 minutes. Animals were given a minimum of 2 minutes rest time between trials.

Fluoro-Jade C Staining

Brains were sectioned at a thickness of 30 μm using a vibratome and the −2 from bregma sections were stained with fluoro-jade C following the manufacture’s protocol (Millipore). Stained sections were imaged at 6 different cortical locations and the number of fluoro-jade positive cells were recorded. The cell count is presented as the number of fluoro-jade positive cells per mm2. The number of cells stained with fluoro-jade within the hippocampus (dentate gyrus, CA1, and CA3) was also recorded and presented as the total number of cells with positive staining.

Paraformaldehyde-fixed primary cortical neurons (3 wells per group) were permeabilized with 0.1% Triton X-100, blocked with 1% bovine serum albumin at 4°C, and then incubated with antibodies against MAP2c (1:100, sc-390543, Santa Cruz Biotechnology) at 4°C overnight. Cells were washed, and incubated with an anti-mouse-Alexa Fluor 546 (1:1000, A11003, Thermo Fisher Scientific) for 1 hr at room temperature. Nuclei were stained with DAPI and washed with PBS. Neurons were then subjected to fluoro-jade C following manufacture’s protocol (Millipore). Seventy-five percent of each well was imaged with an EVOS FL Auto Imaging System (Thermo Fisher Scientific) and then ImageJ multi-point tool was used to count (by an investigator blinded to group assignment) cell number for the green (fluoro-jade C) and blue channels (DAPI staining was used as the total cell count). Replicates were not performed.

Western Blot

Frozen brains (left and right cerebrum without olfactory blubs), placed in Lysis buffer (Biorad), were homogenized and sonicated, and the protein concentration was measured. Cells from neuron cultures (6 wells per group) were sonicated in lysis buffer and the protein concentration was measured. Western blot was performed, as we have previously done (Wu et al., 2018), using primary antibodies for p-AKT (4060S, Cell Signaling Technology), AKT (4691S, Cell Signaling Technology), p-GSK-3β (ab68476, Abcam), GSK-3β (9315, Cell Signaling Technology), BCL2 (2870S, Cell Signaling Technology), CC3 (9661, Cell Signaling Technology), and β-actin (sc-47778, Santa Cruz Biotechnology). For each gel, signals were normalized to β-actin for each lane and then all lanes were normalized to the sham values (2–3 per membrane). Replicates were not performed for the cell culture samples.

Statistical Analysis

Data is presented as individual data points with the mean and standard deviation. All tests were two sided. Data was assessed for normality and homoscedasticity, and if not meet, non-parametric tests were used. A p<0.05 was considered statistically significant. Data for cell counts, western blot, and rotarod were analyzed using one-way ANOVAs with Tukey post-hoc. Neuroscore, beam walking, and T-maze were analyzed using Kruskal-Wallis with Dunn’s post-hoc. GraphPad Prism 6 (La Jolla, CA, USA) and SigmaPlot 11.0 (SysStat, Germany) were used for analyzing and graphing data.

Availability of Data and Material

Data is available upon reasonable request.

Results

Overall, 0/14 sham, 4/17 SAH+Vehicle, 2/16 SAH+Galantamine, 3/16 SAH+Galantamine+MLA, and 2/6 SAH+Galantamine+BAY80-6946 mice died before neurobehavioral testing 1 day post-SAH. Three SAH+Vehicle, 2 SAH+Galantamine, and 2 SAH+Galantamine+MLA mice died before recovering from surgery and so were replaced. Samples from these mortalities were excluded from statistical analysis. Complete Statistical reports are available in the Supplementary Information.

Galantamine Reduces Neuronal Degeneration after SAH

Mice receiving vehicle following SAH had significant neuronal degeneration within the cortex (p=0.0017 vs Sham) and hippocampus (p=0.0096 vs Sham) as indicated by fluoro-jade staining (Figure 1). Treatment with galantamine reduced dystrophic neurons in both brain areas (p>0.05 vs Sham, p<0.05 vs SAH+Vehicle (cortex), p=0.051 vs SAH+Vehicle (hippocampus)). Remarkably, the number of fluoro-jade positive cells in galantamine-treated mice was statistically indistinguishable from sham values. Inhibition of α7-AChR with MLA reversed galantamine’s protective effects on neuronal degeneration (p>0.05 vs SAH+Vehicle, p<0.05 vs Sham (cortex and hippocampus), p<0.05 vs SAH+Galantamine (cortex)).

Fig. 1. Galantamine Reduces Neuronal Cell Death 24 hours post-SAH.

Fig. 1

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a. Representative images showing fluoro-jade positive cells in the cortex of sham mice and SAH mice treated with vehicle, galantamine, and galantamine coadministered with MLA. b. Representative images showing fluoro-jade positive cells in the hippocampus (−2 to −1 bregma; dentate gyrus shown; CA1 also shown in SAH+Galan.+MLA). Scale bars = 100 μm. c. Areas of the cortex used for analysis are shown (boxes). Galantamine significantly reduces the number of fluoro-jade positive cells in the cortex and the hippocampus 24 hour post-SAH. n=4–6/group. * p<0.05 vs Sham, # p<0.05 vs SAH+Vehicle.

Galantamine Improves Functional Performance

One day post-SAH, mice treated with the vehicle had significant neurobehavioral deficits compared to sham mice as indicated by the performances in neuroscore, beam walking, rotarod, and T-maze (p<0.05 vs Sham) (Figure 2). Treatment with galantamine improved mice neurobehavioral performances such that their performance was statistically indistinguishable from sham mice (p>0.05 vs Sham). Galantamine-treated mice showed the most functional improvement on the rotarod test; galatamine-treated SAH mice performed significantly better than vehicle-treated SAH mice (p<0.05). Coadministration of galantamine with MLA prevented galantamine’s beneficial effects.

Fig. 2. Galantamine Improves Functional Outcome 24 hours after SAH.

Fig. 2

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a. Galantamine improves performance on the neuroscore and rotarod tests. Coadministration of MLA reverses galantamine’s protection. * p<0.05 vs Sham, # p<0.05 vs SAH+Vehicle, † p<0.05 vs SAH+Galantamine. n=7–8/group.

Galantamine Promotes Neuronal Survival via α7-AChR Signaling

Effect of α7-AChR

Since galantamine is both an agonist of α7-AChR and a cholinesterase inhibitor, we utilized brain tissue from α7-AChR null mice which were subjected to SAH and then treated with either vehicle (n=4) or galantamine (n=4). Neither vehicle nor galantamine reduced the number of fluro-jade positive degenerating cells in α7-AChR null mice (p<0.05 vs Sham) (Figure 3). In a previous study, galantamine-treated α7-AChR null mice did not have any neurobehavioral improvement, and, in fact, loss of α7-AChR led to worse working memory deficits (Dienel et al., 2021).

Fig. 3. Galantamine is Not Beneficial for α7-AChR Null Mice 24 hours after SAH.

Fig. 3

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a. Representative images of fluoro-jade C staining in the cortex. b. Representative images of fluoro-jade C staining in the hippocampus (−2 to −1 bregma; dentate gyrus shown; CA1 also shown in Sham and SAH+Galantamine). Scale bars = 100 μm. c. Counts of the fluoro-jade C positive cells. n=2–4/group.

α7-AChR Stimulation Reduces Cell Death through Activation of the PI3K/AKT Pathway

PI3K/AKT signaling is a major pathway in promoting cell survival following brain injury. To test the significance of this pathway for α7-AChR agonism after SAH, the PI3K inhibitor, BAY80-6946 was used. BAY80-6946 partially reversed the beneficial effects of galantamine on neurobehavioral tests (Figures 4a–d). For the PI3K/AKT signaling pathway in the SAH injured brain, galantamine promoted neuronal survival after SAH possibly through increasing the expressions of PI3K, p-AKT, and BCL2, while reducing the levels of GSK-3β and CC3. BAY80-6946 partially reversed the elevations of p-AKT and BCL2 provided by galantamine treatment, and completely prevented galantamine’s effect on CC3 levels (Figures 4e–i). Overall schematic of the pathway involved in galantamine’s protection of neuronal apoptosis is presented in Figure 4j.

Fig. 4. Galantamine Signaling Prevents Apoptosis via PI3K/AKT Pathways at 24 Hours after SAH in Mice.

Fig. 4

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a–d. Behavioral performances of SAH mice. n=4–6/group. e–i. Mouse brain protein expressions of PI3K, p-AKT/AKT, p-GSK-3β/GSK-3β, BCL2, and CC3. j. Schematic of galantamine’s mode of action reducing apoptosis. n=4–6/group. * p<0.05 vs Sham, # p<0.05 vs SAH+Vehicle, † p<0.05 vs SAH+Galantamine. Galan: Galantamine. BAY: BAY80-6946.

α7-AChR is highly expressed on neurons and microglia, so to determine if galantamine had a direct effect on neurons, we used primary cortical neuron cultures. Hemoglobin caused a significant increase in the number of fluoro-jade positive neurons compared to saline alone. Galantamine significantly attenuated the number of fluoro-jade positive neurons to a levels which is indistinguishable from saline alone. MLA, a α7-AChR antagonist, reversed galantamine’s protective effects (p=0.0001) (Figures 5a–b). Analysis of protein expression from primary neurons shows that galantamine improved the cell survival (PI3K/AKT) signaling pathway (Figure 5c).

Fig. 5. In Primary Neurons, Galantamine Reduces Apoptosis via PI3K/AKT Pathways at 24 Hours.

Fig. 5

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a. Fluoro-Jade staining of in vitro primary neurons subjected to hemoglobin toxicity. Scale bars = 50 μm Green: fluoro-jade, red: MAP2c, blue: DAPI. b. Analysis of the fluoro-jade positive neurons. n=3 wells/group c. in vitro primary neuron protein expressions after hemoglobin toxicity. n=6 wells/group. * p<0.05 vs Sham, # p<0.05 vs SAH+Vehicle, † p<0.05 vs SAH+Galantamine. Galan: Galantamine. BAY: BAY80-6946. Hb: Hemoglobin.

Discussion

Previous studies have suggested that α7-AChRs are a treatment target for brain hemorrhages. Herein, we examined the potential of α7-AChR as a therapeutic target for SAH. We observed that galantamine, an FDA-approved cholinergic drug with α7-AChR agonist properties, reduced neuronal dystrophy and improved neurobehavioral outcomes after SAH in mice. In mice lacking α7-AChR (i.e. α7-AChR null mice), galantamine did not have any beneficial effect on either neuronal degeneration or behavior. In fact, α7-AChR null mice with SAH had more degenerating neurons than wild-type mice following SAH. Using primary neuron cell culture, we observed that α7-AChR agonism with galantamine provided a direct reduction in neuronal dystrophy after hemoglobin toxicity. Finally, the PI3K/Akt signaling pathway is responsible for promoting neuronal survival by α7-AChR agonism after SAH. The findings of this study support the potential of α7-AChR agonism as a treatment target for SAH.

Early brain injury, and more specifically neuronal apoptosis has been suggested to be a contributing factor of delayed cerebral ischemia following SAH (Macdonald, 2014). As such, preventing neuronal apoptosis may provide a therapeutic benefit, which is supported by experimental studies (Croci et al., 2021; Duris et al., 2011; Zhou et al., 2021). Clinical trials for early brain injury have begun to gain traction with the understanding that early brain injury may be a treatable phenomenon. Since the α7-AChR agonist galantamine is FDA-approved, it may be a good candidate for clinical trials.

In the present study, mice with SAH have a significant number of degenerating neurons in the cortex and hippocampus which contributes to the poor overall functional performance one day after SAH. Administration of galantamine was able to significantly reduce the number of degenerating neurons in both the cortex and hippocampus, thereby improving functional outcome. Our findings of α7-AChR agonism providing improved neuronal survival after SAH support the findings of others (Duris et al., 2011). Furthermore, the Western blot results indicate that galantamine and α7-AChR agonism promotes cell survival via the anti-apoptotic PI3K/Akt signaling pathway, which has been suggested by others (Duris et al., 2011; Krafft et al., 2012).

Galantamine has several mechanisms, such as agonism of α7-AChR and antagonism of acetylcholinesterase. Furthermore, α7-AChR is known to have high expression on microglia and neurons in rodent brains, so there may be mechanisms in both cell types which contribute to the overall protection against SAH injury. In order to determine if α7-AChR agonism by galantamine causes a direct signaling for neuronal protection, primary cortical neuronal cell culture was used. The findings indicate that α7-AChR agonism can promote neuronal cell survival after hemoglobin toxicity. Use of the α7-AChR antagonist MLA in the presence of galantamine further supported our interpretation of the neuronal cell culture data. Western blot analysis of the proteins from the neuronal cell cultures indicated that the PI3K/AKT signaling pathway is stimulated, thereby reducing the expression of cleaved caspase 3.

Further support for the neuroprotective role of α7-AChR comes from the use of α7-AChR knockout mice. Mice lacking α7-AChR were unable to receive a therapeutic benefit from galantamine treatment; galantamined-treated α7-AChR knockout mice with SAH had similar numbers of degenerating neurons as vehicle-treated α7-AChR knockout mice with SAH. Although not powered to test for significant differences in neuronal degeneration between α7-AChR knockout and wild-type SAH mice, a sample size estimation indicates that six mice per group are needed (see Supplementary Information), which suggests that there may be a significant differences in the number of degenerating cells in the cortex after SAH (i.e. α7-AChR knockout mice have more degenerating cortical cells than wild-type mice). This is important evidence that α7-AChR is a crucial receptor in outcome after SAH. Future studies are needed to fully understand the impact of α7-AChR in SAH outcome, including the use of α7-AChR knockout mice.

Study Limitations

A number of limitations exist for this study. First, only one α7-AChR agonist at a single dose was used. Other α7-AChR agonists have been beneficial for brain injury. We chose galantamine because it is used in humans and the dose was determined from human doses (Zhao et al., 2018) which was adjusted for intranasal administration. Second, females nor aged mice were used in this study. In a previous study, female mice treated with galantamine had improved functional outcome compared to vehicle-treated female mice (Dienel et al., 2021). However, the neuronal survival in female mice treated with galantamine was not assessed. Whether α7-AChR agonism is beneficial in aged mice with SAH remains to be studied. Third, since α7-AChR agonism can provide beneficial via microglia (i.e. anti-inflammatory) or neurons (i.e. cell survival), the benefits observed in mice may be the combined benefit of anti-inflammation and neuroprotection. Indeed, in a previous study we observed a protective benefit observed by galantamine in microglia cell culture (Dienel et al., 2021). Another limitation is that only the number of α7-AChR knockout mice used was low. Due to the low number of mice used, we did not perform any statistical analysis. Future studies will be performed to better understand how knockout of α7-AChR affects SAH outcome. Finally, we only examined cell survival at one day post-SAH. Future studies are needed to understand cell survival on other days during the first week after SAH since α7-AChR agonism may delay cell death rather than preventing it. Previous data from a 28-day study of α7-AChR agonism by galantamine indicates less white matter damage in galantamine-treated mice compared to vehicle-treated mice on day 28 (Dienel et al., 2021). The data from that study suggests that cell death is prevented, however future studies are needed to confirm this.

Supplementary Material

1

Highlights.

  • α7-AChR agonism reduces neuronal apoptosis after SAH, thereby improving functional

  • outcome

  • Knockout of α7-AChR does not improve outcome after SAH

  • The anti-apoptotic mechanism of α7-AChR agonism is via PI3K/Akt signaling in neurons

Acknowledgements

Funding support was provided by the Brain Aneurysm Foundation (D.W.M), a National Institute of Health R01 (D.W.M.) and K23 (S.L.B.), Center for Clinical and Translational Sciences Scholar Program (H.A.C.), and a seed grant provided by the Department of Neurosurgery at UTHealth (D.W.M).

Footnotes

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Conflicts of Interest

H.A.C. is involved in a clinical trial (NCT02872857). All other authors declare no conflicts of interest.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1723379-supplement-1.pdf (255.5KB, pdf)

Data Availability Statement

Data is available upon reasonable request.

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