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. Author manuscript; available in PMC: 2018 May 22.
Published in final edited form as: Neurosci Lett. 2017 Apr 2;650:18–24. doi: 10.1016/j.neulet.2017.03.053

Brain injury results in lower levels of melatonin receptors subtypes MT1 and MT2

Nicole D Osier a,b, Lan Pham a, Bunny J Pugh b, Ava Puccio a,c, Dianxu Ren a, Yvette P Conley a,d, Sheila Alexander a,e, C Edward Dixon b,c,f,*
PMCID: PMC5886725  NIHMSID: NIHMS866252  PMID: 28377323

Abstract

Background

Traumatic brain injury (TBI) is a devastating and costly acquired condition that affects individuals of all ages, races, and geographies via a number of mechanisms. The effects of TBI on melatonin receptors remains unknown.

Purpose

The purpose of this study is to explore whether endogenous changes in two melatonin receptor subtypes (MT1 and MT2) occur after experimental TBI.

Sample

A total of 25 adult male Sprague Dawley rats were used with 6 or 7 rats per group.

Methods

Rats were randomly assigned to receive either TBI modeled using controlled cortical impact or sham surgery and to be sacrificed at either 6- or 24- hours post-operatively. Brains were harvested, dissected, and flash frozen until whole cell lysates were prepared, and the supernatant fluid aliquoted and used for western blotting. Primary antibodies were used to probe for melatonin receptors (MT1 and MT2), and beta actin for a loading control. ImageJ and Image Lab software were used to quantify the data which was analyzed using t-tests to compare means.

Results

Melatonin receptors levels were reduced in a brain region- and time point-dependent manner. Both MT1 and MT2 were reduced in the frontal cortex at 24 hours and in the hippocampus at both 6 hours and 24 hours.

Discussion

MT1 and MT2 are less abundant after injury, which may alter response to MEL therapy. Studies characterizing MT1 and MT2 after TBI are needed, including exploration of the time course and regional patterns, replication in diverse samples, and use of additional variables, especially sleep-related outcomes.

Conclusion

TBI in rats resulted in lower levels of MT1 and MT2; replication of these findings is necessary as is evaluation of the consequences of lower receptor levels.

Keywords: Traumatic brain injury (TBI), brain trauma, controlled cortical impact (CCI), rat, melatonin, receptors

Introduction

TBI is a devastating condition that globally affects individuals at all stages of life [1,2]. In the United States of America (USA) alone, a recent estimate values the direct and indirect costs of TBI at a staggering $76.5 billion annually [3]. Unfortunately, acute and chronic disability remains common [4]. No therapy has demonstrated sufficient safety and efficacy to warrant translation to TBI clinical care [5]. Thus, the quest to identify effective therapies for TBI remains a worldwide initiative. Many major barriers to identification of new effective therapies exist. For example, TBI is characterized by a wide variety of cellular and histopathological changes [6], suggesting drugs with multiple mechanisms of action or a combination of therapies may be necessary [7]. Also, in order for a TBI therapeutic to be effective, it must be able to reach and exert its effects in the brain; however, many pharmaceutical compounds lack the necessary properties (e.g. small; lipophilic) to efficiently cross the blood-brain-barrier [8] via natural (i.e. unassisted) mechanisms.

One promising potential TBI therapeutic is melatonin (MEL), which readily and rapidly crosses the blood-brain-barrier [9]. MEL is produced throughout the body, with the primary site of production being the pineal gland. MEL is available as a medication and over-the-counter supplement and has a known low toxicity profile in both human and animal studies with few reported adverse effects even at very high doses [10]. Existing evidence shows endogenous MEL levels are altered in TBI-survivors in a time-point and biosample-dependent manner [11,12]; thus, MEL may be important in the body’s response to TBI and there may be an opportunity to improve outcomes via therapeutic administration of MEL. Pre-clinical studies have found MEL leads to attenuation of one or more of the histopathological and functional consequences of TBI [13,14]. Moreover, published evidence suggests that MEL has many mechanisms of action, including anti-apoptotic [15,16], anti-oxidative [17], and anti-mitophagic [18] properties; notably, the abovementioned pathways are all well-established as implicated in TBI-pathology.

Despite the beneficial characteristics of MEL, current evidence remains largely limited to a small number of preclinical studies which yielded conflicting evidence. More concerning, none of the studies to date have confirmed that a major target for MEL therapy (MEL-specific receptors, MT1 and MT2) remains unaltered by TBI. This pilot study is the first to explore MT1 and MT2 levels after TBI, thereby addressing a major gap in the knowledge. Hypothesis: decreased levels of MEL-specific receptors (MT1 and MT2) may occur in response to TBI. Lower levels of MEL receptors have previously been reported in an animal model of depression [19] and following treatment with the melatonin receptor antagonist luzindole [20]. In this study western blot was used to semi-quantify melatonin-specific receptor levels (MT1 and MT2) within the hippocampus and frontal cortex during the acute (6 hr and 24 hr) period post-TBI.

Materials and Methods

Methods Overview

All experimental procedures were approved by the Institutional Animal Care & Use Committee prior to beginning study activities. Prior to the enrollment, and throughout the duration of the study, test animals were kept in a climate-controlled housing facility on a 12 hour light/dark cycle. Rats were randomly assigned to be subjected to TBI using the controlled cortical impact (CCI) model or sham surgery and then be humanely euthanized at either 6 hr or 24 hr after surgery. Brain tissue was harvested, the ipsilateral (i.e. injured) hippocampus and frontal cortex dissected, and flash frozen; whole cell lysates were stored at −80 °C until later processed for whole cell lysates and used for western blotting.

Sample

In this pilot study, the sample was comprised of male Sprague Dawley rats (Harlan, Indianapolis, IN, USA); at the time of surgery all test animals were young adults (10–14 week old), weighing 275–375g. The rationale for the chosen sample demographics was to control for the confounding effects of age, brain development, and sex on TBI outcomes [21–24]. In total, 25 rats were included in this exploratory pilot study, resulting in a 6 or 7 rats per group across the 4 groups: (1) CCI with 6 hr sacrifice (n= 6), (2) sham with 6 hr sacrifice (n= 6), (3) CCI with 24 hr sacrifice (n= 6), and (4) sham with 24 hr sacrifice (n= 7).

Surgery

Prior to surgery, the CCI device was examined and test fired to ensure proper functioning (e.g. the piston fires freely). Rats received inhaled anesthetic immediately prior to and throughout surgery. Each rat was placed in an anesthesia induction chamber and given 4.0% isoflurane in a 2:1 mixture of N2O:O2. Once sedated, the rat was intubated and placed into a stereotaxic frame, secured using bilateral ear bars and a single incisor bar. Isoflurane levels were reduced to a maintenance dose (2.0%) throughout the surgery, unless the rat showed signs of regaining consciousness, in which case the dose was increased. The head was shaved with electric trimmers and the surgical site prepared using betadine and sterile gauze. A scalpel was used to make a midline incision approximately 20mm in length. The muscles were gently separated and the skin and fascia reflected using sterile surgical tools and cotton-tipped applicators. A pneumatic drill was used to make a craniectomy on the exposed skull between the lambda and bregma (anterior-to-posterior) and also between the coronal ridge and sagittal suture (medial-to-lateral). The window was approximately 7 mm, just large enough for unobstructed clearance of the 6 mm tip. The detached bone flap was carefully removed using microdissecting forceps so as to not breach the dura and subsequently discarded.

The piston was gently lowered to ensure that it was centered within the bone window and to confirm unobstructed clearance for the 6 mm diameter rigid, flat-beveled tip. The device was zeroed to the cortical surface and gently withdrawn to avoid surgical site disruption. The piston assembly was adjusted to reflect the desired impact parameters: depth of 2.8 mm, velocity of 4 m/s, and dwell time (i.e. duration) of 150 ms. At this point the device was actuated to induce TBI. In both CCI- and sham-exposed rats, the surgical site was sutured closed, topical anesthetic applied, and anesthesia discontinued. The animal was removed from the stereotaxic frame, extubated, and assessed for righting reflex. Following return of spontaneous locomotion, regular housing and husbandry were resumed. Animals were monitored for evidence of pain and distress and analgesic was administered per institutional protocol. Sham control rats received identical surgical and post-surgical treatment to TBI-exposed animals but were not be exposed to CCI.

Sacrifice

Animals were humanely euthanized at one of two post-surgery time points: 6 hr or 24 hr. At the time of sacrifice, animals were injected with Fatal Plus (0.25 mL per rat) and decapitated by guillotine. Brains were rapidly harvested and the ipsilateral fontal cortex and hippocampus dissected over ice, placed in microcentrifuge tubes, and flash frozen in liquid nitrogen. Tubes containing dissected tissue were stored at −80° C until processed for analysis.

Tissue Processing

A lysis buffer was prepared, composed of: 0.01M Tris-Cl/0.1M NaCl, 0.001M ethylenediaminetetraacetic acid (EDTA), 1 μg/mL aprotinin, and 100 μg/mL phenylmethylsulfonyl fluoride (PMSF). Specific volumes of lysis buffer were pipetted onto the brain tissues (200 μL for frontal cortex; 100 μL for hippocampus). A sonicator was used to homogenize the tissue and generate whole cell lysates, which were centrifuged at high speed in a cold (4°) room for 30 minutes. Following separation of the layers, the supernatant fluid was collected into a microcentrifuge tube, vortexed to homogenize, and aliquoted out into smaller tubes to minimize the effects of freeze/thaw cycles.

BCA Assay

On the day the gel was to subject to electrophoresis, the protein content of the samples was determined using a Pierce bicinchoninic acid (BCA) assay (Thermo Fisher Waltham, MA, USA). Samples were diluted five-fold and loaded in duplicate into a 96 well plate. For comparison, 8 standards of known protein concentration were loaded in triplicate. A spectrophotometer (Molecular Devices, Sunnyvale, CA, USA), and associated Softmax Pro software (Molecular Devices, Sunnyvale, CA, USA) were used to determine the volume of supernatant fluid needed to load a consistent mass of 20 μg total protein per well.

Sample Preparation and Wet Laboratory Methods

Samples were prepared by combining the volume of sample required for the desired mass of protein, with Bolt™ Sample Reducing Agent (Thermo Fisher Scientific, Waltham, MA, USA), and Bolt™ LDS Sample Buffer (Thermo Fisher Scientific, Waltham, MA, USA). The mixture was centrifuged briefly before boiling for 10 minutes; boiled samples were allowed to cool and were re-centrifuged at room temperature. Prepared samples were loaded into a Bolt™ 4–12% Bis Tris Plus 15 well gel (Thermo Fisher Scientific, Waltham, MA, USA) along with a SeeBlue® Plus2 Pre-stained Protein Standard ladder (Thermo Fisher Scientific, Waltham, MA, USA). The gel was electrophoresed at a constant 165 volts for approximately 30 minutes.

A first generation Invitrogen Bolt™ semi-dry transfer system (Thermo Fisher Scientific, Waltham, MA, USA) was used in accordance with the manufacturer’s instructions orienting the anode stack on the bottom, the polyvinylidene fluoride (PVDF) membrane in the middle, and the cathode stack on top. The transfer program was run for a total semi-dry transfer time of 7 minutes. Immediately following transfer, the gel was retrieved and placed in a tray and a small volume (~5 mL) of GelCode™ Blue Stain Reagent (Thermo Fisher Scientific, Waltham, MA, USA) was poured over the gel and allowed to incubate on a rocker for at least 1 hour to ensure that there were no issues during electrophoresis.

Next, membranes were labeled, rinsed with deionized water, and then rinsed with methanol. Membranes were then washed in Tris-Buffered Saline and Tween 20 (TBS-T) for 3 washes of 5 minutes each. Next, membranes were blocked for 30 minutes in 5% blotting-grade non-fat dry milk (BioRad, Hercules, California, USA) and then incubated overnight in 5% milk with the primary antibodies as described in additional detail below. Following incubation with the primary antibody, membranes were re-washed for 15 minutes (3 washes at 5 minutes each) in TBS-T. Membranes were then incubated in 1% milk with the corresponding secondary antibody described below. A commercially available (Super Signal West Femto Maximum Sensitivity Substrate) two-component chemiluminescent solution (Thermo Fisher Scientific, Waltham, MA, USA) was applied to the membrane (total volume = 1.0 mL per membrane, with the two parts in equal volume). The membrane was imaged using a digital imager (BioRad, Hercules, California, USA). Following imaging, and prior to repeating the blocking, staining, and imaging steps for the remaining antibodies, the membranes were washed (15 minutes, as before), stripped with Restore™ PLUS Western Blot Stripping Buffer (Thermo Fisher Scientific, Waltham, MA, USA) for 15 minutes and rewashed (15 minutes, as before).

First, membranes were probed for MT1 (ab184013, 1:1000, Abcam, Cambridge, UK) with goat-anti-rabbit secondary antibody (#31460, 1:5000, Thermo Scientific, Waltham, MA, USA). Second, membranes were probed for MT2 (ab203346, 1:1000, Abcam, Cambridge, UK) with (#31460, 1:5000, Thermo Scientific, Waltham, MA, USA) goat-anti-rabbit secondary antibody. Finally, membranes were probed for beta actin (a2066, 1:2500, Sigma Aldrich, St. Louis, MO, USA) with (#31460, 1:5000, Thermo Scientific, Waltham, MA, USA) goat-anti-rabbit secondary antibody, in an effort to control for the possibility of differential protein loading.

Analysis

Image J software (National Institutes of Health, Bethesda, MD, USA) was used in combination with Image Lab software (Bio-Rad, Hercules, CA, USA) to quantify data for analysis. Melatonin receptor levels were normalized to beta actin levels for the same test animal to control for the possibility unequal protein loading. All analysis was conducted using SPSS version 24 (IBM, Armonk, CA, USA) statistical software. Preliminary analysis was completed as follows: t-tests to compare protein levels of sham vs. injured rats at a single time point of either 6 hr or 24 hr post-operatively.

Results

Post-Operative Outcomes

In this study, there was a 0% mortality rate associated with experimental procedures. Moreover, neither sham surgery nor CCI caused significant morbidity (e.g. seizures) that would have necessitated a test animal being prematurely euthanized. Results from western blot analysis are summarized below, with composite gels provided in Figure 1, and a graphical group comparison displayed in Figure 2. The criteria for statistical significance was p < 0.05 (Note: in figure 2, which does not provide exact p-values, statistical significance is denoted as follows: *p < 0.05; ** p < 0.01).

Figure 1.

Figure 1

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Composite figure of western blot results with molecular weight ladder.

Figure 2.

Figure 2

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Graphical comparison of CCI and sham animal western blot results by protein and brain region.

MT1 Levels

When whole cell lysates from ipsilateral frontal cortex of rats exposed to CCI (vs. sham) were compared using western blot, MT1 levels were reduced at 24 hr (p= 0.002), though they were unchanged from sham levels at 6 hr. Moreover, in the hippocampus, MT1 levels were reduced at both 6 hr (p= 0.027) and 24 hr (p= 0.011).

MT2 Levels

As with the cortical MT1, cortical MT2 levels were reduced at 24 hr post-injury (p= 0.010), but unchanged from sham levels at 6 hr. Likewise, in the hippocampus, MT2 levels were reduced at both 6 hr (p= 0.042) and 24 hr (p= 0.001) post-injury.

Actin

In all brain regions and time points examined in this study, there was no statistically significant change in beta actin levels after TBI (compared to sham). This is consistent with what has been reported previously [25–27]. This supports the use of actin to normalize the results of this study in an attempt to control for any inconsistencies in loading samples.

Discussion

Novel Contribution to the Literature and Relationship to Other Published Findings

This study is the first to report a reduction in levels of melatonin receptor subtypes 1 and 2 (i.e. MT1 and MT2) after TBI with evidence of time point- and brain region-specific differences. This reduction in MEL receptors may affect the efficacy of MEL therapy after TBI, though this remains to be empirically tested. Notably, past attempts to treat experimental TBI with MEL therapy have yielded inconsistent results, with many studies showing neuroprotective effects after TBI for at least one of the regimens tested [13,16,28,29], one study showing no effect of therapy [30], and a few studies showing adverse effects of one or more of the therapeutic regimens tested [15,31]. Interestingly, many of the studies that reported beneficial effects of melatonin therapy tested the therapeutic effects in reducing oxidative stress [28,31–36], which rely on melatonin’s receptor-independent free radical scavenging properties, rather than receptor-dependent effects. Thus, the importance of melatonin-specific receptors may have been obscured by studies whose endpoints resulted from receptor-independent activities. Moreover, there is known genetic variation in [37] or near [38] genes encoding MEL receptors, the effect of which was controlled for in this study with the use of congenic (i.e. inbred) rats. Replication in different strains of test animal would strengthen the available evidence, as would clinical studies exploring the effects of MEL receptor polymorphisms on TBI outcomes as well as response to therapy. This emerging line of inquiry may prove relevant for precision therapy initiatives and identification of the subset of patients most likely to benefit from melatonin therapy.

Limitations and Future Directions

All pre-clinical studies have limited clinical applicability and require replication in pre-clinical models before clinical trials can be justified. Importantly, many of the therapies that show success in pre-clinical studies, even when replicated, do not demonstrate therapeutic effects in clinical trials. Moreover, the focal nature of TBI induced using the CCI model means that the results may not hold true when diffuse brain injury and/or polytrauma is present; similarly, these results may not reflect the effects of milder brain injury including closed head injury. Replication using CCI of varying severity along with other injury models (e.g. fluid percussion; blast-induced TBI) is needed. The generalizability of this study is further limited by the homogenous nature of the sample, which was restricted to young adult male rats. Since sex is known to be an important factor in brain trauma [39–41], validation of study findings in female animals is necessary. Likewise, replicating the study using pediatric and aging mice would strengthen the evidence base.

Several specific limitations should be acknowledged and considered when interpreting the results of this pre-clinical study. Western blot analysis of MT1 and MT2 receptor levels provides preliminary evidence of lower numbers of MT1 and MT2 receptors after injury, but additional research using more sophisticated techniques (e.g. immunohistochemistry; gene expression studies) is necessary. Future studies should identify the reason for the reduced number of receptors detectable using western blot after TBI (e.g. Are they being down-regulated? Is the protein itself being damaged/altered by injury?). Moreover, exploring additional time points and brain regions will enhance our understanding of these MEL receptor changes. For example, the exclusion of behavioral endpoints represents a significant limitation in this study and although CCI-exposed animals had lower hippocampal and cortical levels of MT1 and MT2 at 24 hours post-injury, this may not result in changes in symptom profiles.

One important behavioral endpoint worth examining in future studies is sleep. Further inquiry should seek to relate the findings of the present study to sleep-related outcomes after TBI, considering melatonin’s well-established release following diurnal rhythms as well as its’ critical role in maintaining circadian rhythms across the phylogenetic tree [42–49]. Moreover, the relationship between MT1 and MT2 protein expression/levels in biological fluids and circadian rhythms as well as sleep-wake cycles has also been reported [50–52] as have the utility of melatonin receptor agonists at improving sleep-related outcomes [53–57]. Taken together, existing evidence suggests that the findings of reduced abundance of melatonin receptors after TBI in this study may alter sleep-related outcomes that were unstudied in the present pilot project. This is especially of interest, consider TBI is known to result in altered circadian rhythms [58] as well as sleep-related problems including but not limited to insomnia, hypersomnia, altered sleep timing, difficulty maintaining sleep, sleep-disordered breathing, and nightmares [12,59–66] that may be due in part to changes within the melatonergic system, especially the receptors, that are yet to be characterized.

There are also some practical limitations of this study that should be acknowledged. For example, whole cell lysates were generated, the supernatant fluid collected, and the resulting pellet disposed of. Thus, it was not possible to use tissue subcellular fractionation techniques with differential centrifugation to evaluate the membrane and nuclear MEL receptors in isolation [67]. A related consideration is that while the use of actin to normalize comparisons of melatonin receptor concentrations between sham and injured animals is consistent with most western blot research, the method has limitations. Specifically, there can be regional changes in the ratio of cell types that could confound the interpretation of findings in this and other studies. For example, in a situation characterized by neuronal death accompanied by increased gliosis, actin levels in total protein would appear unchanged, but fail to account for the loss of cells that would otherwise express MT1 and/or MT2 within injured brain regions. Not only would this obscure the ability to accurately interpret the cause of melatonin receptor loss, it would also complicate attempts to therapeutically target these changes.

Conclusions

This study is the first to demonstrate that MEL receptors are affected by TBI. Specifically, time point- and region-specific decreases in both MT1 and MT2 levels occurred after TBI. Replication of these results is necessary using more diverse pre-clinical samples (e.g. other strains/species, females, older/younger animals) and studies with additional cellular and behavioral endpoints. Clinical research exploring the effects of TBI on MEL receptors and trialing the effects of therapeutic MEL may be warranted. Overall, the results of this study, along with the existing literature, suggest the melatonergic system is implicated in TBI pathology and/or recovery and is worth further study.

Acknowledgments

This work was supported by the National Institutes of Health [T32NR009759, F31NR014957]; The Copeland Award of The Pittsburgh Foundation; the International Society of Nurses in Genetics; The Neurotrauma Nursing Foundation/American Association of Neuroscience Nursing; and Sigma Theta Tau International Eta Chapter. In addition, we would like to thank Michael Farmer for his assistance with the figures.

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