Neuroprotective Effect of Enriched Chicken Bone Broth as a Dietary Supplement in a Model of Migraine Mediated by Early Life Stress

Abstract

Early life stress is a risk factor for development of migraine, a prevalent painful neurological disease characterized by sensitization and activation of trigeminal neurons. Secondary early life stress was previously shown to cause increased expression of neuronal proteins implicated in peripheral and central sensitization. Recently, dietary supplementation of chicken bone broth was shown to attenuate trigeminal nociception in an orofacial pain model. Accordingly, the focus of this study was to determine the effects of early life stress and dietary inclusion of bone broth on trigeminal nociceptor sensitization and activation in a model of episodic migraine. Adult Sprague-Dawley male sender rats subjected to primary traumatic stress were placed next to breeding or pregnant female rats that served as receiver rats (secondary traumatic stress) and in proximity to the offspring until weaning. Unstressed and stressed young adult offspring were tested for mechanical nocifensive response after exposure to a pungent odor known to be a migraine trigger, and in response to daily supplementation of bone broth. Early life stress promoted a primed state of trigeminal nociceptors that were activated by the pungent odor in both genders. Female animals exhibited a higher basal sensitization level and prolonged nociception compared with males. Supplementation of bone broth beginning at the time of weaning inhibited basal and triggered trigeminal mechanical sensitivity. Early life stress caused development of a sensitized trigeminal system that is implicated in migraine pathology and dietary supplementation with bone broth suppressed trigeminal sensitization, and thus may provide neuroprotective activity for reducing migraine risk.

Introduction

Migraine is a chronic neurological disease involving intense headaches and autonomic symptoms such as photophobia and phonophobia.^1,2 Migraine is most prevalent in women and is associated with considerable individual, family, and societal burden.^3–5 A key feature of the migraine phenotype is a state of trigeminal sensitization that lowers the activation threshold of nociceptive neurons to peripheral stimuli.⁶ Many risk factors, including stress, anxiety, and sleep deprivation, are thought to promote a persistent state of central sensitization and increased sensitivity in migraine patients.^7,8 Unmanaged daily stress is implicated in migraine pathology; however, there is evidence that early life stress should also be considered a risk factor for migraine later in life.^9–11

Recently, we demonstrated that early life stress was associated with elevated levels of several signaling proteins in trigeminal neurons known to promote peripheral and central sensitization.¹² A sensitized trigeminal system would increase the excitability state of nociceptive neurons and thus decrease the amount of stimulus required for activation. Interestingly, many of the known triggers of migraine are physical stimuli such as flickering lights, loud or irregular sounds, or pungent odors, including the California bay laurel tree, also referred to as the headache tree.^13–15

Umbellulone, a volatile leaf compound, causes activation of trigeminal neurons through activation of the transient receptor potential ankyrin 1 (TRPA1) receptor and subsequent release of calcitonin gene-related peptide from primary trigeminal nociceptive neurons.¹⁴ Exposure of sensitized animals to an extract of the bay leaf caused an increase in the number of nocifensive responses of trigeminal neurons to mechanical stimulation in a model of episodic migraine that was inhibited by noninvasive vagus nerve stimulation.¹⁶

Orofacial pain conditions, including migraine and temporomandibular disorders (TMDs), involve sensitization and activation of trigeminal neurons that provide a pathway for pain transmission.^17,18 The three branches of the trigeminal nerve, ophthalmic (V1), maxillary (V2), and mandibular (V3), which provide sensory innervation of the head and face, are responsive to thermal, mechanical, and chemical stimuli.¹⁹ In a recent study, dietary inclusion of an enriched chicken bone broth (ECBB) before prolonged jaw opening, which was used as a model of TMD pathology, significantly inhibited V3 nocifensive response to mechanical stimulation.²⁰

The enriched bone broth contained polyphenolic compounds with antioxidant properties and selectively inhibited cyclooxygenase (COX)-2 activity, a key enzyme involved in synthesis of prostaglandins that mediate inflammation and nociceptor sensitization and are the primary target of nonsteroidal anti-inflammatory drugs (NSAIDs).²¹ In addition, dietary inclusion of the bone broth significantly inhibited stimulated expression of protein kinase A in neurons and glial cells within the upper spinal cord. Taken together, these findings support the notion that ECBB can modulate the trigeminal system to reduce nociception and central sensitization in a model of TMD.

Migraine, another prevalent orofacial pain disease, is often comorbid with TMD in susceptible individuals since both involve central and trigeminal sensitization.²² Therefore, a main goal of this study was to determine if dietary supplementation with ECBB would function in a protective manner to inhibit trigeminal activation in a model of episodic migraine induced by early life secondary stress.

Materials and Methods

Animals and experimental model

All animal care and protocols were approved by Missouri State University's Institutional Animal Care and Use Committee (IACUC ID: 17-010.0). In addition, an effort was made to reduce the number of animals in this study and to limit their suffering. In this study, a total of 39 male and 45 female Sprague-Dawley animals were utilized, with a similar number of animals included in each of the experimental conditions.

Early life secondary traumatic stress was induced similarly to a recently described method.¹² In brief, nonbreeding male rats (senders) were subjected to the Morris water maze for 3 min or until finding an underwater platform. The animal was then removed and allowed to rest for 2 min before being placed back in the water tank. At the conclusion of this stressful event, which was repeated four times in total, the animal was returned to its cage and then placed between an age-matched male and female rat, which were designated as receiver animals. This stress procedure was repeated every day for 7 days at three specific times that included the time immediately preceding breeding, at the start of gestation, and before weaning (Fig. 1).

FIG. 1.

Experimental timeline.

A breeding pair not directly subjected to the primary stressor was housed in separate cages on either side of the swimmer rat at all times, except when the two rats were cohoused to breed in the week after the sender rat's first week of stress exposure. The sender rat was exposed to Morris water maze swimming during the week before mating of the parents, the week after copulation of the breeding pair, and the week before weaning of the offspring (postnatal days 14–21). The same timeline was followed for a naive control group that was housed in a separate, but identical room, and not in the presence of a primary stressed sender rat.

From day 21 onward (weaning), offspring with early life stress were provided either water (control), or 1% (w/w) ECBB (kindly provided by International Dehydrated Food, Inc.) that was utilized in a previous study involving trigeminal nociceptor activation in a model of TMD pathology.²⁰ In brief, the ECBB was prepared in water with no additives from finely ground raw chicken bones under high temperature and pressure for a minimum of 8 h, and after removal of fats was evaporated and then spray dried.

The dry product comprised 95% protein, 0% fat, and 5% ash, which contained the minerals calcium, phosphorous, magnesium, and potassium (personal communication with International Dehydrated Foods, Inc.). The majority of protein in the broth was hydrolyzed collagen, mostly in the form of type II collagen, and also contained chondroitin sulfate. Body weight and food and liquid consumption were monitored weekly and no marked differences were observed between any of the groups.

Assessment of mechanical sensitivity

After 4 weeks of feeding, mechanical nociception in two orofacial regions was determined in offspring following an established protocol.^16,20 Before testing, animals were acclimated to restraint in the Durham Animal Holder (Ugo Basile, Varese, Italy) for 5 min on 3 consecutive days. During this time, animals were conditioned to a mechanical stimulus by gently rubbing the hair follicles and cutaneous region over the temporalis and along the masseter.

After acclimations, animals were allowed to rest for 2 days before behavior testing. Mechanical nocifensive thresholds were determined in response to a series of calibrated von Frey filaments (Stoelting, Wood Dale, IL, USA) applied in increasing force to the cutaneous tissue over the temporalis (V1) and masseter muscle (V3). A researcher who was blinded to experimental conditions determined a positive response, which was defined by head withdrawal before the bending of the filament, and the response was recorded by a second researcher.

Each filament was applied five times on each side of the face, and data were reported as the average number of head withdrawal responses obtained from five applications of each specific calibrated filament. The 60 and 100 g filament were chosen as the filaments of interest for the V1 and V3 regions, respectively, since basal positive responses to this force were consistently less than one bilaterally, whereas the 100 and 180 g filaments regularly caused more than three out of five responses per side.

Exposure to reported migraine trigger

To determine if early life stress could function as a risk factor to lower the activation threshold of trigeminal neurons to mechanical stimulation, some animals were exposed to a pungent odor known to cause activation of sensitized trigeminal neurons in an animal model of episodic migraine.¹⁶ Animals were exposed for 10 min to a pungent odor from an oil extract of California bay laurel leaves (CBL) by placing 20 μL of the oil on a cotton swab and placing the animal in a chamber with an oxygen flow of 2 L/min. The cotton swab was positioned so that the animal could not come into direct contact with the oil but would be subjected to the oil's volatile compounds, including the TRPA1 receptor activating molecule umbellulone.¹⁴

Mechanical nociception testing was performed before CBL exposure to determine basal levels and then again at 2, 24, and 48 h postexposure to determine the temporal response, which mimics the duration of episodic migraine.²³ Animals were sacrificed after all testing was complete by CO₂ asphyxiation and decapitation.

Statistical analysis

Behavioral data were analyzed using SPSS 24 software (IBM, North Castle, NY, USA). Owing to non-normal distribution of data, nonparametric Mann–Whitney U tests were performed for pairwise comparisons between groups at each time point. Two measurements were considered significantly different when P < .05.

Results

Early life stress promotes trigeminal sensitization in offspring: inhibition by dietary bone broth

Changes in trigeminal nociception of female and male rats exposed to early life secondary traumatic stress were determined in response to mechanical stimulation of two common orofacial regions reported by migraine patients during an attack, the area over the temporalis (V1) and masseter (V3). Results are presented as the average number ± standard error of the mean (SEM) of nocifensive head withdrawals in response to stimulation of the V1 region with the 60 g von Frey filament and the V3 region with the 100 g filament.

As seen in Figure 2, female offspring exposed to early life stress exhibited an increase in the average number of basal nocifensive withdrawal responses in both V1 and V3 regions when compared with naive control, but only the V3 value was significantly elevated over naive levels (P = .010). Inclusion of ECBB as a dietary supplement resulted in a decrease in the basal level of sensitization of both V1 and V3 trigeminal neurons when compared with levels in the stressed female animals, being significantly different in the V3 region (P = .003).

FIG. 2.

Early life stress promotes an increase in nociception in V3 trigeminal neurons in female offspring that is inhibited by inclusion of ECBB. Results are shown for unstimulated female naive animals, animals exposed to secondary stress and receiving only water (stressed), or animals exposed to secondary stress and receiving dietary supplementation of ECBB. The average number ± SEM of head withdrawals to mechanical stimulation of V1 (top panel) or V3 (bottom panel) neurons is shown. A significant increase from the naive animals is indicated by an asterisk, whereas a significant decrease from stressed animals is indicated by a pound sign. ECBB, enriched chicken bone broth; SEM, standard error of the mean.

In male animals the average nocifensive response was lower than females for all conditions, and no significant differences were observed between the average number of withdrawals in response to mechanical stimulation of V1 or V3 neurons of stressed or ECBB animals when compared with naive values (Fig. 3). However, stressed female animals (Fig. 2) exhibited a significantly higher basal level of nociception in V1 (P = .026) and V3 (P < .001) regions when compared with stressed male offspring (Fig. 3).

FIG. 3.

Early life stress does not result in a significant change in nocifensive response in V1 or V3 trigeminal neurons in male offspring. Results are shown for unstimulated male naive animals, animals exposed to secondary stress (stressed), or animals exposed to secondary stress and receiving dietary supplementation of ECBB. The average number of head withdrawal responses ± SEM to mechanical stimulation of V1 (top panel) or V3 (bottom panel) neurons is reported.

Exposure to pungent odor triggers enhanced nociception in male and female offspring: inhibition by dietary bone broth

Trigeminal nocifensive response to mechanical stimulation was measured in female and male animals initially exposed to early life secondary stress and then 2 h after exposure to the pungent volatile compounds from an oil extract of CBL, which is a known migraine trigger in susceptible individuals.¹⁴ The average number of nocifensive responses was significantly increased in female stressed offspring 2 h after exposure to the CBL extract when compared with levels in naive females exposed to CBL in both the V1 (P = .028) and V3 regions (P = .041) (Fig. 4).

FIG. 4.

Exposure to pungent odor causes a sustained increase in trigeminal nociception in female offspring animals subjected to early life stress that was inhibited by dietary bone broth. Results are shown for unstimulated female naive animals, animals exposed to secondary stress and receiving only water (stressed), or animals exposed to secondary stress and receiving dietary supplementation of ECBB. The average number ± SEM of head withdrawals to mechanical stimulation of V1 (top panel) or V3 (bottom panel) neurons is shown. A significant increase from the naive animals is indicated by an asterisk, whereas a significant decrease from stressed animals is indicated by a pound sign.

The average number of nocifensive responses in V1 and V3 trigeminal neurons remained elevated compared with naive levels at the 24 and 48 h time points. Inclusion of ECBB before CBL was sufficient to inhibit the level of nociception in stressed females to levels observed in naive animals at each of the time points. Significant differences in nociceptive responses between female stressed and ECBB animals was seen at 2 h (P = .022) and 48 h (P = .008) in the V1 neurons and 24 h after CBL exposure in V3 neurons (P = .014).

As seen in Figure 5, the average number of nocifensive responses was significantly increased in both V1 (P = .012) and V3 (P = .001) neurons in male offspring exposed to secondary early life stress 2 h post-CBL when compared with naive males. In contrast to female stressed animals (Fig. 4), the average number of nocifensive responses to mechanical stimulation returned to near naive levels 24 and 48 h after CBL exposure. Dietary supplementation with ECBB significantly inhibited nociception of V3 neurons (P = .029) but did not reach significance in the V1 region.

FIG. 5.

Exposure to pungent odor mediated a transient elevation in trigeminal nociception in male offspring animals subjected to secondary early life stress that was inhibited by dietary bone broth. Results are shown for unstimulated male naive animals, animals exposed to secondary stress and receiving only water (stressed), or animals exposed to secondary stress and receiving dietary supplementation of ECBB. The average number ± SEM of head withdrawals to mechanical stimulation of V1 (top panel) or V3 (bottom panel) neurons is shown. A significant increase from the naive animals is indicated by an asterisk, whereas a significant decrease from stressed animals is indicated by a pound sign.

Discussion

Sensitization, or a lowering of the activation threshold of trigeminal neurons, is implicated in the underlying pathology of migraine and other orofacial pain conditions, such as TMD, that are more prevalent in females than males.^22,24,25 In this study, the hypothesis that exposure to secondary traumatic stress during early development would lead to enhanced basal nocifensive head withdrawal response to mechanical stimulation of the V1 and V3 trigeminal regions was investigated. The rationale for this study was based on recently published findings that early life stress caused a sustained elevation in the levels of several key proinflammatory and pronociceptive signaling proteins in the trigeminal ganglion and spinal trigeminal nucleus in the offspring.¹²

Elevated levels of mitogen-activated protein kinases in these regions are implicated in the initiation and maintenance of peripheral and central sensitization,²⁶ which are physiological changes in the nervous system involving enhanced communication between neurons and glial cells.^27,28 Migraine and TMD pathology are associated with peripheral and central sensitization that can progress to a primed state of both primary and secondary nociceptive neurons through prolonged cellular changes in the expression of ion channels, receptors, and signaling pathways.²⁹

Results from this study provide evidence that secondary early life stress promotes an increase in the average number of nocifensive responses in the V1 (temporalis) and V3 (masseter) region in female animals when compared with male offspring. Taken together, exposure to early life secondary stress is likely to mediate cellular changes in trigeminal neurons and glia to promote a sustained higher state of basal mechanical sensitivity in female offspring.

To determine whether early life stress could promote a sensitized state of trigeminal nociceptors that functions as a risk factor for migraine, an episodic migraine animal model involving trigeminal nociceptor activation in response to exposure to a pungent odor reported to be a migraine trigger in humans was utilized.¹⁶ Both male and female offspring exposed to early life stress exhibited an increase in the average number of nocifensive responses to mechanical stimulation of the V1 and V3 regions 2 h postexposure to the migraine trigger. Interestingly, although the average number of nocifensive responses returned to basal levels in male animals 24 h after the odorant trigger, female animals continued to exhibit higher nocifensive responses in V1 and V3 regions at 24 and 48 h.

These findings support the notion that early life stress may function as a migraine risk factor by lowering the activation threshold of trigeminal neurons indicative of a primed nociceptive state that underlies chronic pain conditions and is more severe in females.²⁹ The enhanced mechanical sensitivity in the temporalis and masseter muscles may be clinically relevant since migraineurs often report increased pain (hyperalgesia) as well as allodynia, a painful response to a normally nonpainful stimulus, in their head and face.^30,31 Furthermore, our findings are in agreement with data from human studies that early life stress is associated with an increased risk of migraine and other orofacial pain conditions, and support the notion that female gender should be considered a risk factor associated with lower activation thresholds of trigeminal nociceptive neurons.^9,10,32

In this study, dietary supplementation with an ECBB was sufficient to mediate a decrease in the basal level of sensitivity in female animals and suppressed trigeminal nociceptor activation in response to the migraine trigger, an extract of California bay leaves, in both males and females. This finding is in agreement with results from a prior study in which daily inclusion in the diet of an ECBB inhibited trigeminal activation in a model of TMD, which involves activation of V3 trigeminal neurons, caused by prolonged jaw opening.²⁰ The rationale for including the bone broth before an activating stimulus is that dietary supplements have traditionally been used to maintain homeostasis/health and prevent a pathological event rather than acting as an abortive therapy.

Findings from our studies provide evidence that dietary inclusion of ECBB mediates a neuroprotective role in a model of episodic migraine and TMD pathology. Although not a focus of this study, the ability of ECBB to prevent trigeminal pain signaling is likely to be meditated by its antioxidant activity/reducing potential and its ability to inhibit synthesis of proinflammatory prostaglandins through selective inhibition of COX-2 but not COX-1.²⁰ Inhibition of COX activity is a characteristic of NSAIDs, which are effective in reducing pain levels in migraine and other orofacial pain conditions.^33,34

In addition, the bone broth was found to inhibit the neuronal upregulation of protein kinase A, a signaling protein known to promote peripheral and central sensitization in trigeminal nociceptive neurons.³⁵ The benefit of the ECBB may also involve the anti-inflammatory and antioxidant activity reported for hydrolyzed collagen,³⁶ collagen peptides,³⁷ and chondroitin sulfate,³⁸ which are all components of this broth.

Importantly, inflammation and oxidative stress are implicated in the underlying pathology of migraine.³⁹ Thus, it is possible that each of these properties of the bone broth may be partly responsible for the observed neuroprotective effect in the episodic migraine and TMD models. However, it is also plausible that the antinociceptive effect of the bone broth may be due, at least in part, to its ability to influence the gut microbiota to favor bacteria that produce anti-inflammatory molecules. There is a growing body of literature to support the notion that a healthy nervous system is linked to a healthy gut.^40–42

Furthermore, the results with respect to the beneficial properties of bone broth to inhibit trigeminal nociceptor activation is consistent with the reported inhibitory effects mediated by dietary inclusion of cocoa^43,44 and grape seed extract.⁴⁵ Given the opioid epidemic and our overdependence on all types of drugs to relieve pain, the identification of novel nonpharmacological therapeutic strategies to inhibit pain pathways and minimize unwanted negative side effects is greatly needed. Taken together, our findings have demonstrated in preclinical models that dietary inclusion of food products provides a novel nonpharmacological method for modulating the trigeminal system and, therefore, may be beneficial in reducing the risk of experiencing migraine or TMD later in life.

Ethics approval

All animal care and protocols were approved by Missouri State University's Institutional Animal Care and Use Committee (IACUC ID: 17-010.0).

Authors' Contributions

O.J.P. was responsible for breeding animals and the generation, analysis, and presentation of much of the behavioral data and assisted in drafting the original article. L.E.C. was primarily responsible for the collection and analysis of the behavioral results and assisted in the writing and editing of the final article. P.L.D. was responsible for the study design and final analysis of the results as well as writing and editing.

Author Disclosure Statement

P.L.D. received grant support for this study from International Dehydrated Foods, Inc.

Funding Information

Funding and the enriched chicken bone broth product utilized in this study were provided by International Dehydrated Foods, Inc.