Abstract
There is a great need for effective treatment options for post-traumatic stress disorder (PTSD). Neuropeptide Y (NPY) is associated with resilience to traumatic stress. MC4R antagonists, such as HS014, also reduce response to stress. Both regulate stress-responsive systems – the hypothalamic–pituitary–axis (HPA) and the noradrenergic nervous system and their associated behaviors. Therefore, we examined if their intranasal delivery to brain could attenuate development of PTSD-related symptoms in single prolonged stress (SPS) rodent PTSD model. Three regimens were used: (1) prophylactic treatment 30 min before SPS stressors, (2) early intervention right after SPS stressors, (3) therapeutic treatment when PTSD behaviors are manifested 1 wk or more after the traumatic stress. NPY delivered by regimen 1 or 2 prevented SPS-triggered elevation in anxiety, depressive-like behavior, and hyperarousal and reduced dysregulation of HPA axis. Hypothalamic CRH mRNA and GR in ventral hippocampus were significantly induced in vehicle- but not NPY-treated group. NPY also prevented hypersensitivity of LC/NE system to novel mild stressor and induction of CRH in amygdala. Some of these impairments were also reduced with HS014, alone or together with NPY. When given after symptoms were manifested (regiment 3), NPY attenuated anxiety and depressive behaviors. This demonstrates strong preclinical proof of concept for intranasal NPY, and perhaps MC4R antagonists, for non-invasive early pharmacological interventions for PTSD and comorbid disorders and possibly also as therapeutic strategy.
Introduction
As exposure to life-threatening traumatic stress is an inherent and often unavoidable aspect of military operations, stress-triggered neuropsychiatric disorders, especially post-traumatic stress disorder (PTSD), are a very serious problem for the armed forces. PTSD is a severe debilitating neuropsychiatric disorder that develops in a subset of individuals following a life-threatening trauma. Longitudinal studies of PTSD epidemiology found that 17% of all patients who reported exposure to traumatic events are ultimately diagnosed with PTSD (using DSM-5 criteria) 12 mo after the traumatic event.1
PTSD is characterized by intrusive recollections of the traumatic event, avoidance of reminders of the event, hyperarousal or hypervigilance, and negative alterations in cognition and mood associated with the traumatic event. It is often comorbid with depression and other anxiety disorders, as well as with substance abuse, sleep disturbances, and marked psychosocial and occupational impairments. Dysregulation of the hypothalamic–pituitary–adrenocortical (HPA) axis and increased activity of central and peripheral noradrenergic system are usually observed in PTSD patients.2–4
Treatment of PTSD is extremely challenging with a few effective options. Standard treatments include mostly symptomatic medications and cognitive behavioral therapy. The only two medications approved by the FDA for treatment of PTSD are the selective serotonin reuptake inhibitors (SSRIs), sertraline and paroxetine. However, as many as 40–50% of patients do not respond to current treatments and develop a chronic course of the illness. Network meta-analysis of pooled data revealed that the medications used have only moderate strength of evidence supporting their effectiveness.5 Improved therapies are urgently needed for treatment as well as for prevention of PTSD. Such therapies could save billions of dollars in medical care and in reduced productivity and could provide enormous societal benefits.
Extensive and compelling evidence demonstrated that neuropeptide Y (NPY) can mediate resilience or recovery from harmful effects of stress and may be protective against developing stress-related disorders such as PTSD and comorbid depression [reviewed in6–12]. Genetic studies in humans and rodents found that lower NPY levels are associated with more anxiety and higher reactivity to emotional and stressful challenges.9,13,14 In military survival training, soldiers with higher NPY were found “stress hardy” with positive coping.15 Significantly lower plasma and CSF concentrations of NPY were found in individuals with combat-related PTSD than in control subjects or combat-exposed non-PTSD group and were inversely correlated with the severity of the disease.16–18 Administration of NPY to brain by intracerebroventricular (icv) infusion or microinjections to selective brain regions in animal models provided resilience to a number of stress-triggered impairments.19–22
Although less well studied than NPY, there is evidence that the melanocortin subtype 4 receptor (MC4R) is involved in stress-induced changes in behavioral responses and stress-related disorders. For example, the selective melanocortin MC4R antagonists, HS014, MCL0020, or MCL0129, given to rats by icv infusion shortly before restraint or immobilization reduced stress-elicited anorexia and depressive-like behavior and reduced learned helplessness.23 Conversely, icv infusion of the MC4R agonist α-MSH induced robust activation of the HPA axis activity following a stressful event, suggesting that these receptors may be involved in the control of the HPA axis functions during stress.24 Several studies demonstrated an interaction between the NPY and the melanocortin system, leading us to speculate that there may be synergistic effects between them.25,26
Therefore, our recent work aimed to see if NPY and/or a MC4R antagonist could be delivered in a non-invasive way to prevent or treat the development of PTSD and comorbid disorders. Intranasal infusion is increasingly being appreciated as a non-invasive mechanism to bypass the blood–brain barrier.27 Here, we provide a summary of our previously published findings with intranasal infusion of neuropeptide Y and/or HS014, a MC4R antagonist, to prevent or treat PTSD-associated symptoms in a rodent model of PTSD.11,28–34 The appropriateness for translation into the military and issues that still remain to be resolved are discussed.
Methods
All experiments were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee at NYMC and the USAMRMC Animal Care and Use Review Office. Male Sprague–Dawley rats (7 wk of age, 150–160 g) from Charles River (Wilmington, MA, USA) were housed in a barrier area on 12 h light/dark cycle at 23 ± 2°C with ad libitum access to food and water. After a 14-d acclimation period, rats (at least eight per group) were randomly assigned to the experimental or control groups.
Animals were exposed to single prolonged stress (SPS) animal model of PTSD, with strong face and construct validity35,36 with some modifications.29 The SPS procedure was performed between 9 a.m. and 2 p.m. Rats were immobilized for 2 hours on a metal board by taping the limbs with a surgical tape and restricting the motion of the head. Following immobilization, they were immediately subjected to the forced swim for 20 min in a plexiglass cylinder (50 cm height, 24 cm diameter; Stoelting, Wood Dale, IL, USA) filled to two-thirds with 24°C fresh water. The animals were dried and allowed to recuperate for 15 min and then exposed to ether vapor until loss of consciousness.
Intranasal NPY was administered in three regimens: (1) prophylactic treatment 30 min before SPS stressors under isoflurane anesthesia, (2) early intervention or secondary treatment right after exposure to the SPS stressors while still under the influence of ether, (3) therapeutic treatment after symptoms manifested 7 d or more after exposure to SPS under isoflurane anesthesia. HS014 was administered in the first two regimens, either alone or with low concentration of NPY. For intranasal administration, NPY and/or HS014 was freshly dissolved in distilled water and 10 μL was carefully infused into each nostril with pipettman and disposable plastic tip as described previously.29 Animals were compared with those identically treated but receiving equal volume of intranasal vehicle or to unstressed controls.
In some experiments, short-term changes evaluated. Otherwise, animals were left undisturbed for at least a week before behavioral or biochemical assays. Anxiety was determined on elevated plus maze and open-field, depressive-like behavior by forced swim test, and hyperarousal by acoustic startle response. Following sacrifice, brains were processed for immunocytochemistry (tyrosine hydroxylase, glucocorticoid receptor [GR], or corticotropin-releasing hormone [CRH] or selective regions [locus coeruleus (LC), ventral and dorsal hippocampus, and mediobasal hypothalamus] dissected, to determine changes in mRNA levels for selective genes of interest by qRT-PCR or protein by immunoblots. In some experiments, plasma ACTH and corticosterone were determined by ELISA.29
Results
Intranasal administration to rats of NPY and/or HS014 enabled delivery to the brain and showed that they can rapidly modify behavior.28,29 Fluorescent NPY reached widespread regions of the brain within 30 min following intranasal infusion.11,30
As shown in Figure 1, three regimens were used to examine how they might prevent the development or treat PTSD symptoms: (1) prophylactic treatment 30 min before SPS stressors, (2) early intervention right after SPS stressors, and (3) therapeutic treatment when PTSD behaviors manifested (>1 wk after stressors).
Effects of traumatic stress of SPS on the development of PTSD-associated symptoms and intervention with intranasal infusion. In the absence of intervention, traumatic stress of SPS leads to development of PTSD-associated symptoms and impairments in behavioral, neuroendocrine, and biochemical features, which are evident 7 d or later afterwards. Interventions with intranasal NPY and/or HS014 were applied for (1) prophylactic treatment 30 min before SPS stressors, (2) early intervention or secondary treatment right after exposure to the SPS stressors, and (3) therapeutic treatment after symptoms manifested. (Note. SPS, single prolonged stress; HPA, hypothalamic–pituitary–adrenal axis; LC/NE, locus coeruleus noradrenergic system; CRH, corticotropin-releasing hormone; GR, glucocorticoid receptor).
A week or more after SPS stressors, the animals displayed behavioral, neuroendocrine, and biochemical symptoms (Fig. 1). Animals given vehicle had elevated anxiety, depressive-like behavior, and hyperarousal. In contrast, these behaviors were similar to unstressed controls with early intervention or prophylactic NPY treatment (regimen 1 or 2) with intranasal NPY (100 μg/rat). The side effects were minimal. Intranasal NPY did not change the stress-triggered effects on body weight29 and had a very transient effect on feeding behavior (unpublished results). There were no major side effects on the cardiovascular system, with possible benefit from transient amelioration of the SPS-triggered elevation in heart rate.37
When given near the exposure to SPS stressors (regimens 1 and 2), intranasal NPY also prevented dysregulation of HPA axis likely by restoring proper negative feedback inhibition via functional GR. With early intervention, hypothalamic CRH mRNA and GR, as well as phosphorylated GR in ventral hippocampus, were significantly higher in vehicle- but not in NPY-treated group.30 NPY also prevented SPS-triggered sensitivity of central noradrenergic system to novel mild stressor and induction of CRH in amygdala.34
Some of these impairments were also reduced with intranasal HS014, alone or together with NPY. Administration of the MC4R antagonist, HS014 in regimen 1, provided prophylactic treatment against development of anxiety (tested on elevated plus maze) at a wide range of concentrations (3.5 ng or 100 μg/rat) and depressive-like behavior at high concentrations (100 μg/rat).28 This was accompanied by inhibition of SPS-elicited induction of CRH gene expression in the PVN and increased levels of GR in the hippocampus.33 Moreover, low concentrations of NPY (50 μg/rat) and HS014, (3.5 ng/mL), which by themselves were ineffective, prevented depressive-like behaviors when combined.38 With early intervention (regimen 2), HS014 (3.5 ng or 100 μg/rat) could reduce development of depressive-like behavior (or re-experiencing), but not anxiety symptoms.38
Intranasal NPY administered when SPS-triggered symptoms are already manifested (regimen 3) reduced features of anxiety and depression. Thus, when given a week after SPS stressors, intranasal NPY (150 μg/rat) led to reduced anxiety and immobility on forced swim test 2 d later.32
Discussion
The findings suggest that intranasal NPY has a great promise for early intervention or secondary treatment to prevent the development of PTSD. This now needs to be translated to human studies. The studies established that a single dose of intranasal NPY when given immediately after the traumatic stress of SPS had long-lasting beneficial effects. The potential for early intervention was also shown when injection of NPY into the hippocampus 1 hour after the exposure to stress in the predator stress rodent PTSD model prevented the development of behavioral and neurochemical impairments.19
One of the attractive features of intranasal administration of NPY is its ease of application, even self-administration, thus making it a feasible treatment for warfighters even in a remote setting. It could also be a part of the medic’s kit to administer to warriors recently subjected to life-threatening traumatic combat stress.
Our studies showed protective effects of intranasal NPY when given 30 min before the traumatic stress. Although longer times before the stress were not yet examined, if the protection is sustained, intranasal NPY might provide promising non-invasive prophylactic treatment for individuals likely to be exposed to traumatic stress, such as early responders, warfighters, and other military personnel.
Further work needs to be done to determine the therapeutic practicality of MC4R antagonists, either alone or in conjunction with NPY. The results indicate that addition to NPY infusion of a MCR4 antagonist, such as HS014, could enable effective secondary treatment at lower dose than NPY alone. To our knowledge, this is the first study to administer intranasal HS014. However, recently, intranasal HS014 was reported to also block withdrawal hyperalgesia in alcohol-dependent rats.39 Although HS014 is a modified peptide, there are many other MC4R antagonists described,23 which might be more stable and should be tested for their ability to intervene in preventing or treating PTSD or enhance the effects of NPY.
It remains to be determined whether treatment with intranasal NPY can become a feasible approach once the symptoms are manifested. A small phase 1 human clinical trial in patients with PTSD (Clinical trial.gov, ID: NCT01533519) has yielded encouraging preliminary results.40 Additional clinical and preclinical studies will be a key to help clarify the extent and duration of the improvements and the response to multiple treatments.
Overall, the research provides strong preclinical proof of concept for intranasal NPY and perhaps MC4R antagonists as promising non-invasive early pharmacological interventions to prevent development of PTSD and comorbid disorders and possibly also as a therapeutic strategy.
Acknowledgments
We gratefully acknowledge the talented post-doctoral fellows and students who contributed to this work, especially Andrej Tillinger, Marcela Laukova, Lishay Alaluf, and Robert Camp as well as Maria Gullinello, Albert Einstein College of Medicine, for initial consultation with the behavioral tests.
Presentations
Presented as a poster at the 2016 Military Health System Research Symposium (abstract number: 16-0696).
Funding
This work was supported by the Office of Assistant Secretary of Defense for Health Affairs, Medical Research under Award Numbers W81XWH-11-2-0090 and W81XWH-16-1-0016.
References
Author notes
The views expressed in this article are those of the authors and do not necessarily represent the official position or policy of the U.S. Government, the Department of Defenses, or the Department of the Army.
