Acute Dose-Dependent Neuroprotective Effects Of Poly(pro-17βestradiol) in A Mouse Model Of Spinal Contusion Injury

Abstract

17β-Estradiol (E2) confers neuroprotection in preclinical models of spinal cord injury when administered systemically. The goal of this study was to apply E2 locally to the injured spinal cord for a sustained duration using poly(pro-E2) film biomaterials. 

As people learn more about spinal cord injuries, we are beginning to realize the impact it has on people's physical and mental health. Although spinal cord injuries can cause many adverse effects, fortunately, their impact on memory can be controlled.

First, we need to clarify what spinal cord injury is. The spinal cord is a nerve protected in the spine that is responsible for transmitting instructions from the brain to various parts of the body. If the spinal cord is injured, some parts of the body will lose good connection with the brain, resulting in affected limb movement, sensation, and autonomic control.

Spinal cord injuries may have a certain impact on people's memory, especially in the early stages of rehabilitation of patients with spinal cord injuries. Spinal cord injuries may cause a person's attention, working memory, and emotional processing ability to deteriorate, which will affect a person's memory ability.

However, these negative effects will gradually decrease as patients undergo rehabilitation treatment. Patients can improve their memory by participating in memory games and other cognitive training. At the same time, physical rehabilitation is also conducive to restoring the body's motor and sensory functions, thereby improving emotional and cognitive functions.

We should encourage patients with spinal cord injuries to actively participate in rehabilitation treatment and persist in rehabilitation training to improve physical and mental health. Please believe that with the help of correct cognition and postoperative treatment, patients can overcome the impact of spinal cord injury on memory and continue to live an active life. It can be seen that we need to improve memory, and Cistanche can significantly improve memory because Cistanche can also regulate the balance of neurotransmitters, such as increasing the levels of acetylcholine and growth factors, which are very important for memory and learning. In addition, Cistanche can also improve blood flow and promote oxygen delivery, which can ensure that the brain obtains sufficient nutrition and energy, thereby improving brain vitality and endurance.

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Following contusive spinal cord injury in adult male mice, poly(pro-E2) films were implanted subdurally, and neuroprotection was assessed using immunohistochemistry 7 days after injury and implantation. In these studies, poly(pro-E2) films modestly improved neuroprotection without affecting the inflammatory response when compared to the injured controls. 

To increase the E2 dose released, bolus-releasing poly(pro-E2) films were fabricated by incorporating unbound E2 into the poly(proE2) films. 

However, compared to the injured controls, bolus-releasing poly(pro-E2) films did not significantly enhance neuroprotection or limit inflammation at either 7 or 21 days post-injury. Future work will focus on developing poly(pro-E2) biomaterials capable of more precisely releasing therapeutic doses of E2.

INTRODUCTION

Traumatic spinal cord injury (SCI) is a debilitating condition affecting over 18,000 people every year in the United States,1 while the global prevalence of SCI is estimated to be between 236 and 1009 per million.2 

 Patients affected by SCI incur severe neurological deficits including loss of sensation, impaired bowel/bladder function, and paralysis.3,4 The primary impact on the spinal cord results in neuronal death at the injury site which is further exacerbated by several secondary injury cascades involving astrocytes and macrophages. 

Over time, these astrocytes form a glial scar which prevents the extension of axons beyond the lesion edge.5 There are no FDA-approved neuroprotective drugs to date for treating SCI. 

Research spanning several decades has focused on systemic administration of biomolecules to prevent neuronal loss due to secondary injury and/or to promote axonal regeneration, ultimately leading to restoration of lost function.6 One biomolecule that has demonstrated significant promise in preclinical models of SCI is the female sex hormone, 17β-estradiol (E2).

E2 administration is broadly beneficial to the central nervous system by reducing inflammation,7–9 alleviating glial cell reactivity,10 limiting oxidative stress,11–14 and mitigating glutamate-induced excitotoxic neuronal death.15 E2 improves functional recovery in various rodent models of SCI.10,16,17 Interestingly, multiple-dose systemic administration of E2 induces functional recovery more quickly18,19 compared to animals receiving only a single dose.17 

Since E2 promotes functional recovery more effectively through multiple-dose regimens, our goal was to stably release E2 locally at the injury site (to prevent any off-target effects) to circumvent problems arising from multiple dosages/systemic injections of E2. 

To that end, we employed a poly(prodrug) biomaterial strategy in which the native drug (E2) was first functionalized with reactive allyl groups and then polymerized via photoinitiated thiol–ene radical addition with a flexible oligo-(ethylene glycol) dithiol, as previously described.20 We obtained high molecular weight polymer chains, which consist of an E2 prodrug as the repeating unit within the polymer. 

Upon hydrolytic degradation, the polymer gradually releases E2 into the surrounding media. The only other byproducts from the release are carbon dioxide and oligo(ethylene glycol) (oEG) (Figure 1). Poly(pro-E2) chains fabricated into films or microfibers can release E2 at nanomolar concentrations in vitro on a time scale of months to years.20 

The E2 released from these poly(pro-E2) scaffolds is bioactive, and these scaffolds promote neurotrophism and neuroprotection in vitro by protecting neurons from hydrogen peroxide-induced oxidative stress. Here, we applied the poly(pro-E2) films in a murine model of contusion SCI, immediately following injury, and assessed their neuroprotective capability in vivo. 

Our initial poly(pro-E2) film design increased neuroprotection acutely (7 days post-injury) at the injury site without affecting the inflammatory response. Since poly(pro-E2) films release nanograms of E2 per day, we developed another release strategy where unbound E2 was incorporated into the poly(pro-E2) matrix. We hypothesized that bolus release of unbound E2 may provide additional neuroprotective benefits. 

Poly(pro-E2) films released E2 in a burst manner followed by sustained release of E2 from the polymer at lower concentrations. Immunohistological staining assessed neurons, astrocytes, and macrophages/ microglia present at the injury site acutely (7 days post-injury) and chronically (21 days post-injury) after SCI in the presence of these bolus-releasing poly(pro-E2) films.

RESULTS AND DISCUSSION

Poly(pro-E2) films release E2 at nanomolar concentrations, promote neuronal outgrowth from the dorsal root ganglion, and protect neurons from hydrogen peroxide-induced oxidative stress in vitro. 20 The study presented here seeks to determine the ability of poly(pro-E2) films to provide neuroprotection in vivo using a murine model of contusion SCI. 

A moderate contusion SCI was induced in adult male mice, and immediately following the injury, poly(pro-E2) films were placed directly above the injury (Figure 2A). To ensure the poly(pro-E2) films remained in place following implantation, poly(pro-E2) films loaded with rhodamine B were implanted into the spinal cords of injured animals. Seven days post-implantation, spinal cords were collected and imaged using a light microscope (Figure 2B). In all animals, the implanted rhodamine B-loaded poly(pro-E2) films remained in place validating the implantation protocol. 

To test the ability of poly(pro-E2) films to provide neuroprotection and alleviate inflammation, poly(pro-E2) films (without rhodamine B) were implanted following a spinal contusion injury, and the spinal cords were collected 7 days after injury and implantation. Longitudinal sections (10 μm thick) were cut, centered around the injury epicenter, and immunolabeled to visualize neurons and macrophages in the spinal cord (Figure 3). 

Sections were labeled for NeuN to assess the neuronal presence, and quantitative analysis of the staining revealed that the poly(pro-E2) films significantly increased the NeuN positive area around the injury epicenter compared to the injured controls that did not receive film implants (Figure 3A; p = 0.0002). Additional staining for axons using NFH (neurofilament heavy chain) also revealed the acute neuroprotective effect of the implanted poly(pro-E2) films (Figure 3B; p = 0.0017). 

We observed that this change in neuroprotection was not due to a change in the apoptosis (caspase 3) at the injury site (Supporting Figure S1; p = 0.9966). Staining for activated macrophages (CD68) also did not reveal any significant differences between the poly(pro-E2) film-treated animals and the untreated animals (Figure 3C; p = 0.7207). 

Taken together, poly(pro-E2) films modestly increase neuroprotection acutely without altering the inflammatory response. Poly(pro-E2) films increased neuronal density acutely by almost 20%, but the films had no obvious anti-inflammatory effects (based on the quantification of the area occupied by the activated macrophages) (Figure 3). 

This lack of a reduction in inflammation may indicate that the anti-inflammatory and neuroprotective doses of E2 are distinct and that both were not achieved with the current film formulation.20 We hypothesized that increasing E2 release from the poly(pro-E2) films acutely may reduce inflammation and enable a more robust neuroprotective response. 

To increase the dose of E2 released acutely, the poly(pro-E2) films were modified so that bolus E2 release would occur immediately after implantation. After the bolus release of E2, polymer degradation would follow to release E2 at the same concentration as before. The experiment described above was repeated using these bolus-releasing poly(pro-E2) films, and neuroprotection and inflammation were assessed at 7 days post-injury (dpi). 

However, instead of an additional benefit, the bolusreleasing poly(pro-E2) films did not significantly increase the NeuN positive area compared to the injured, untreated controls (Figure 4A; p = 0.6770). The bolus-releasing poly(proE2) films increased CD68 expression, but the increase was not statistically different (p = 0.1780). 

Increasing trends in glial reactivity and microglial inflammation were also observed in the presence of the bolus-releasing poly(pro-E2) films due to the increase in GFAP (astrocytes) and Iba1 (microglia) expression; however, these increases were not statistically greater than what was observed in the injured, untreated controls (p = 0.3233 and p = 0.3053, respectively). Taken together, bolus-releasing poly(pro-E2) films did not enhance neuroprotection, and astrocyte reactivity and macrophage/microglia presence were not reduced as initially hypothesized.

Since differences between injured, untreated animals and injured, bolus-releasing poly(proE2) film-treated animals were not observed at the acute, 7 dpi time point, a chronic time point was selected for further investigation since the poly(pro-E2) films can release E2 long-term.20 The experiment described above was repeated with the bolus-releasing poly(pro-E2) films, but the animals were assessed chronically at 21 dpi (Figure 5). 

Spinal cords were labeled for the same cell types stated earlier. Following analysis, there was a trend toward an increase in the area fraction of NeuN in the bolus-releasing poly(pro-E2) film-implanted animals compared to the injured, untreated animals (Figure 5A; p = 0.0897). In addition, trends were observed where bolus-releasing poly(pro-E2) films decreased the area fraction of CD68 and GFAP expression compared to data from untreated, injured animal controls (Figure 5B, C; p = 0.0859 and p = 0.2780). 

However, none of the trends were statistically different. On the other hand, Iba1 labeled tissue sections from bolus-releasing poly(pro-E2) treated animals displayed increased expression compared to data from untreated, injured controls (p < 0.0001). Our previous work shows that poly(pro-E2) films cast on 15 mm × 15 mm coverslips release about 266 ng of E2 per day,20 which translates to about 2 ng of E2 per day from the films (1 mm × 1.8 mm) used here. The bolus-releasing poly(pro-E2) films release an additional 40 μg of unbound E2 acutely following implantation. 

Research focusing on the use of E2 in rodent SCI models shows that low-dose estrogen (10 μg/kg) administered intravenously attenuates reactive gliosis and provides neuroprotection while attenuating inflammatory events, inhibiting apoptosis, and increasing angiogenesis.21 Conversely, high, systemic doses of estrogen have adverse effects which include increased rates of deep vein thrombosis and cancer22,23 and the development of feminine physical traits in males. This is noteworthy since most of the E2 research has focused on delivering E2 systemically either intravenously or intraperitoneally. 

Considering the safety concerns with the use of high doses of E2 systemically, it is important to optimize E2 concentration for local delivery to the injured spinal cord to prevent side effects. To date, only one study has delivered E2 from a biomaterial by using nanoparticles to focally deliver E2 (2.5 μg or 25 μg). Release from nanoparticles rapidly decreased inflammation acutely in a rat model of contusion SCI.9 The lack of a statistically significant response in our data (Figures 4 and 5) may be due to an imbalance in the amount of E2 that we are delivering locally to the spinal cord with too much E2 (40 μg) being released acutely while very little (2 ng) is being released chronically. 

Although what we observe from these data are modest trends, this study highlights the importance of E2 dosing to promote neuroprotection in vivo. Reducing the initial dosage of E2 and adjusting the degradation kinetics of the poly(pro-E2) biomaterial could help alleviate this problem and potentially provide better neuroprotective outcomes. 

Although this study was performed solely in males, it is crucial to assess the effect of these poly(pro-E2) films in both sexes to establish a valid translational potential for this biomaterial. Research has shown that following SCI, increased estrogen levels in females may be partially responsible for their improved functional outcomes compared to males.24 Since there is an increased estrogen presence in females, any biomaterial strategy using estrogen has to take this increased level into account and modify the material release kinetics accordingly to ensure that the dosage delivered remains the same for males and females.

METHODS

Poly(pro-E2) Films Fabrication.

The poly(pro-E2) polymer P1 was synthesized and characterized as described in detail previously.20 The polymer has a weight-average molecular weight of MW ~ 80 kDa and a dispersity of Đ ~ 3, according to gel permeation chromatography (GPC). Poly(pro-E2) films were fabricated by dissolving the polymer in chloroform (5 wt %/wt; polymer/ chloroform), drop-casting the solution onto 15 mm × 15 mm glass coverslips (100 μL solution per coverslip), and leaving them to dry overnight under vacuum (<100 mTorr) at room temperature (RT). In a subset of experiments, the polymer solution was supplemented with rhodamine B (0.005 wt %/wt; rhodamine B/chloroform) (Sigma-Aldrich, St. Louis, MO) to verify the presence of the poly(pro-E2) films in animals post-implantation.
To create poly(pro-E2) films with unbound E2, films were fabricated by adding unbound E2 (Abcam, Cambridge, MA) to the polymer solution (0.0625 wt %/wt; E2/chloroform) before drop-casting the solution onto the coverslips. Before implantation, the coverslips with films were sterilized using ultraviolet (UV) radiation for 30 min and the films were peeled from the coverslips in a sterile biosafety cabinet. The poly(pro-E2) films were then cut into 1 mm × 1.8 mm pieces, suitable for implantation in an adult mouse as described below.

Contusion Spinal Cord Injury and Poly(pro-E2) Films Implantation.

All surgical and postoperative care procedures were approved by Rensselaer Polytechnic Institute and The Ohio State University Institutional Animal Care and Use Committees (IACUC). To induce a contusion SCI, adult male C57BL/6 mice (10- to 12-week-old), purchased from Jackson Laboratory (stock no. 000664; Bar Harbor, ME), were anesthetized with an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/kg). The backs of the animals were shaved and aseptically prepared using 70% ethanol and betadine. 

After placement of the animal on a heating pad maintained at 37 °C, the skin and muscle layers were opened to expose the vertebral column, and a dorsal laminectomy was performed on the thoracic ninth (T9) vertebra to expose the spinal cord. A moderate (75 kdyn) contusion SCI was induced at this level using an Infinite Horizons impactor (Precision Systems and Instrumentation, Lexington, KY). 

After hemostasis was achieved, the dura was opened (Figure 2) and poly(pro-E2) film or a bolus-releasing poly(pro-E2) film (1 mm × 1.8 mm) was placed under the dura and above the spinal cord, covering the injury site. In control animals, the dura was opened but the poly(pro-E2) films were not implanted. The muscle layers were then closed using sterile sutures, and the skin was closed using sterile metal wound clips. 

The mice received 2 mL of sterile saline injected subcutaneously and were returned to their cages and placed on a slide warmer set to 37 °C overnight. To ensure proper hydration and to prevent infections, the mice were injected subcutaneously with 1–2 mL of sterile saline and Gentocin (1 mg/kg) daily for the first 5 days post-injury (dpi). Bladders were manually expressed twice a day for the duration of the experiments. All the animals were housed under conventional conditions on a 12-hour light-dark cycle with ad libitum access to food and water.

Tissue Harvesting.

At the designated terminal time points of 7 or 21 dpi, the mice were injected with a lethal dose of ketamine/xylazine (1.5× surgical dose) and transcardially perfused with 0.1 M phosphate-buffered saline (PBS; pH 7.4) followed by 4% paraformaldehyde (PFA; pH 7.4). The spinal cords were removed, post-fixed for 2 h in 4% PFA, and then transferred to 0.2 M phosphate buffer (PB; pH 7.4) overnight. 

The spinal cords were cryoprotected in 30% sucrose for 48 h at 4 °C and then blocked into 10 mm segments centered around the injury site with the dorsal columns facing up. These sections were embedded in a Tissue-Tek optimal cutting temperature medium (VWR International, Radnor, PA) and rapidly frozen on dry ice. All the spinal cords were sectioned at 10 μm on a Microm HM505E cryostat, placed on ColorFrost Plus slides (ThermoFisher Scientific, Waltham, MA), and then stored at −20 °C until used for immunostaining.

Immunohistochemistry and Quantification.

The slides were thawed on a slide warmer for 1 h and then rinsed 3 times in 0.1 M PBS. Endogenous peroxidase activity was quenched by incubating the slides in 6% H2O2 diluted in methanol for 15 min at RT. After washing 3 times in 0.1 M PBS, the sections were incubated with 4% BSA and 0.1% Triton X-100 in 0.1 M PBS for 1 h followed by overnight incubation of the primary antibodies at 4 °C or RT (Table 1). 

The slides were incubated with appropriate biotinylated secondary antibodies (1:1000; Vector Laboratories, Burlingame, CA) at RT for 1 h. To visualize the bound antibody, the slides were incubated with Vectastain Elite-ABC (Vector Laboratories) for 1 h and developed with ImmPACT 3,3′-diaminobenzidine (DAB) or Vector SG substrate (Vector Laboratories) for 10 min. 

The sections were dehydrated through sequential 2 × 2 min incubations in 70% and 90% ethanol, 2 × 3 min in 100% ethanol, followed by 3 × 3 min in Histoclear, and finally coverslipped with Permount (ThermoFisher Scientific). Six sections per animal, spaced 100 μm apart in the injured spinal cord, were used for the quantification analysis. 

The immunolabeled sections were imaged using an Axioplan 2 imaging microscope equipped with an AxioCam digital camera and AxioVision version 4.8.2 software (Carl Zeiss Microscopy GmbH, Jena, Germany) and a 5× objective. 

The number of positive pixels in each of the sections was counted using MIPAR image analysis software (MIPAR, Worthington, OH) to calculate the percent positive area fraction per section. The average percent positive area of the six sections per animal was calculated for each of the stains and reported.

Statistical Analysis.

All data are reported as the mean ± standard deviation. Statistical analysis was performed using the GraphPad Prism 9 Statistical Software (GraphPad Software, San Diego, CA). In all the experiments, 5–6 animals were used per group, originating from at least two different animal lots. To test the differences between the control and poly(pro-E2) film groups, Student's t-test (Figures 3, 4, and 5) was used. Significance was established at p ≤ 0.05.

Supplementary Material

Refer to the Web version on PubMed Central for supplementary material.

ACKNOWLEDGMENTS

The authors thank Wenmin Lai for her assistance with the mouse surgeries.

Funding

This work received support from Craig H. Neilsen Foundation Fellowship (Grant 468116) and Paralyzed Veterans of America Research Foundation Fellowship (Grant 3171) to M.K.G. and from NIH R01 (Grant NS092754) and New York State Spinal Cord Injury Review Board Institutional Support Grant C32245GG to R.J.G. All the animal work was performed with assistance from The Ohio State University Neuroscience Department Surgical Core supported by NINDS Grant P30NS104177.

REFERENCES

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