Blood–Brain Barrier Integrity

Several investigators have hypothesized that alterations in the blood–brain barrier may be caused by exposure to cholinesterase inhibitors such as the insecticides used to treat the military uniforms, the nerve agent prophylactic PB, and possibly sarin, and that such alterations could be enhanced by the stress of deployment (Abdel-Rahman et al., 2002, 2004; Amourette et al., 2009; Friedman et al., 1996; Grauer et al., 2000; Kant et al., 2001; Lallement et al., 1998; Shaikh and Pope, 2003; Shaikh et al., 2003; Sinton et al., 2000; Song et al., 2002, 2004; Tian et al., 2002). Normally the quaternary ammonium structure of PB reduces its ability to enter the brain, precluding its effects on the central nervous system (CNS). Friedman et al. (1996) found that exposing mice to PB accompanied by an inescapable forced swim stress protocol reduced the dose of PB required to produce a 50% inhibition in brain acetylcholinesterase activity to less than one-hundredth of the usual dose, thus supporting the proposed hypotheses. However, numerous subsequent studies using different species (e.g., rats, mice, and guinea pigs) and multiple different stress paradigms have not replicated those initial findings. Rats exposed to pole climbing avoidance stress plus tritiated PB to assess its blood–brain barrier permeability, showed no radioactivity in brain micropunches and cryosections (Amourette et al., 2009). The authors concluded that although PB induced effects on the CNS (based on earlier studies), these effects did not seem to be mediated by a central passage of PB linked to increased permeability of the blood brain barrier caused by stress. Shaikh and Pope (2003) and Shaikh et al. (2003) also found no evidence that stress (forced running) in combination with paraoxon exposure (an organophospshate insecticide that inhibits acetylcholinesterase) enhanced PB uptake into brain in rats. Song et al. (2002, 2004) reported that restraint stress failed to affect uptake of PB into the brain; treatment with paraoxon did compromise the blood–brain barrier, but only in young rats. Tian et al. (2002) reported that neither forced running nor forced swimming had any effect on PB toxicity, PB uptake into brain, or PB-induced brain cholinesterase inhibition in rats. Kant et al. (2001) reported that rats exposed to PB and given foot shock stress did not show enhanced stress effects on performance or in levels of stress hormones, nor did stress enhance the passage of PB into the brain. Furthermore, in three other studies, stress actually mitigated or even precluded uptake of PB into the brains of mice, guinea pigs, and rats (Grauer et al., 2000; Lallement et al., 1998; Sinton et al., 2000).

In contrast, Abdel-Rahman et al. (2002) combined 28 days of restraint stress in rats with dermal exposure to PB, DEET (N,N-diethyl-meta-toluamide), or permethrin, either individually or as a mixture of the three chemicals. The combination of chemicals, but not any chemical or stress alone, disrupted blood–brain barrier integrity and produced neuronal death in the cingulate cortex, dentate gyrus, thalamus, and hypothalamus, but not in other regions of the brain. In a later study by this group (Abdel-Rahman et al., 2004), rats exposed to both stress and the chemicals exhibited decreased brain acetylcholinesterase in the midbrain, brainstem, and cerebellum and decreased m2 muscarinic acetylcholine receptor ligand binding in the midbrain and the cerebellum, areas of the brain where no disruption of the blood–brain barrier was observed. The authors suggested that combined stress and the three specific chemicals can damage the cerebral cortex, hippocampus, and cerebellum even in the absence of apparent blood–brain barrier damage.

Brain Metabolism and Histopathology

Multiple aspects of brain metabolism and associated histopathology have also been studied. Scremin et al. (2005) sought to determine if the exposure of veterans to PB as a protective agent would enhance the response to sarin, and lead to delayed behavioral dysfunction, that is, whether induced neurobehavioral dysfunction outlasted effects on inhibition of cholinesterase. Sprague Dawley rats were exposed to low (subsymptomatic) doses of sarin, with or without PB, for 3 weeks with observations of neurobehavioral symptoms as well as brain metabolism, cerebral blood flow, glucose utilization, heart rate, and locomotor activity carried out at 2, 4, and 16 weeks after exposure. Early changes observed at 2 and 4 weeks postexposure included increased regional cerebral blood flow, a finding consistent with known vascular effects of cholinergic agonists. The results indicate that PB protected against some changes seen in the rats exposed to sarin alone. No symptoms, including any changes in regional cerebral blood flood and glucose metabolism, were present in the animals at 16 weeks postexposure. Thus, these studies do not support the hypothesis that delayed symptoms observed in Gulf War illness could be caused by exposure to PB.

A potential synergism of these chemical exposures was subsequently tested by Buchholz et al. (1997), using lower doses in adult Sprague Dawley rats. CNS uptake of C¹⁴-labeled permethrin in rats that had received a very high dose of PB (7.75 mg/kg) for 10 days was decreased by 30%, while blood levels of permethrin were not altered by PB treatment. The authors concluded that these results were inconsistent with the hypothesized synergy of such compounds in Gulf War illness. Kant et al. (2001) exposed rats to PB alone or in combination with stress (foot shock) and observed that PB decreased blood acetylcholinesterase by half, but had no effect on cortical brain acetylcholinesterase. PB did not further modify blood corticosterone levels elevated by stress. Thus, PB did not exacerbate the effects of stress on either performance measures or on levels of stress hormones.

A mouse model of Gulf War illness was used to study effects on phospholipid metabolism following coadministration of 2mg/kg PB and 200 mg/kg permethrin intraperitoneal for 10 days followed by up to a 150-day observation period (Abdullah et al., 2011). The doses used were equal to the LD₅₀ (lethal dose for 50% of sample cells) for both compounds: the permethrin dose calculated as the highest expected dose of permethrin expected for Gulf War veterans. No effects were observed at 8 days postexposure, but chronic effects, including cognitive impairment and anxiety, as well as increased astrogliosis in the cortex but not the hippocampus, were observed at 150 days; there was no evidence of microgliosis or neuronal loss. Exploratory brain proteomic analysis found alterations in proteins associated with lipid metabolism and molecular transport in the brain, along with endocrine and immune system metabolic changes. In conjunction with the observations of cortical astrogliosis, the findings suggest a persistent and residual adverse effect on the immune system.

In further experiments by the same group, mice exposed to 1.3 mg/kg/day PB orally, plus 0.13 mg/kg/day permethrin and 40 mg/kg/day DEET dermally, and restraint stress for 28 days (Abdullah et al., 2012) were reported to exhibit sensorimotor deficits, anxiety, and increased astrocytosis in the cerebral cortex, but no histopathological consequences in the hippocampus, nor any increase in microgliosis. Brain phosphatidyl choline (PC) and sphingomyelin content were increased, with the increase in PC containing monounsaturated fatty acids being higher than PC with saturated and polyunsaturated fatty acids. The authors interpreted the lipid changes as suggestive of alterations in peroxisomal pathways and stearoyl-CoA desaturase activity that might relate to neurobehavioral and neuropathological symptoms following exposure to Gulf War agents. In further study, it was hypothesized that PB and permethrin would modulate the concentration of PC and sphingomyelin, which are reservoirs required for acetylcholine synthesis, and that this modulation would persist at the chronic (150 days) time point (Abdullah et al., 2013). PC and sphingomyelin were elevated in brains of exposed mice at 150 days after a 10-day exposure. Lysoplatelet-activating factors, which are products of PC, were decreased. Catalase expression (a marker for peroxisomes) was increased in exposed mice. The authors state that these results are similarly suggestive of peroxisomal and lysosomal dysfunction in the mouse brains at chronic time points postexposure.

Husain and Somani (2004) examined exposure to sarin, PB, and PB plus sarin, each with or without exercise, in mice. Exercise was carried out daily for 10 weeks on a treadmill. Daily sarin and PB dosing was administered during the fifth and sixth week only. Biochemical effects were only assayed at 1 day following the 10-week exposures. The exposures, regardless of exercise, significantly lowered butyrylcholinesterase, acetylcholinesterase, and neurotoxic esterase in plasma, platelets, and nerves, as well as the spinal cord, striatum, and cortex. While the authors concluded that exercise enhanced the adverse effects of Gulf War chemical exposures in mice, the effects observed were minimal and not likely to have biological significance.

DEET (40 mg/kg) or permethrin (0.13 mg/kg) or the combination in 70% ethanol was applied daily to adult male rats for 60 days (Abdel-Rahman et al., 2001). All three conditions produced diffuse neuronal cell death in the cerebral cortex, hippocampus, and cerebellum.

Exposure to chlorpyrifos alone or to a combination of PB, permethrin, and chlorpyrifos for 10 days in adult mice both produced pathological changes in hippocampal, cortical, and amygdalar morphometry (Ojo et al., 2014), including an impairment of synaptic integrity in the hippocampus and altered neuronal differentiation in the dentate gyrus at 3 days postexposure. Both exposures also increased astrocytic glial fibrillary acidic protein immunoreactivity in the piriform cortex, motor cortex, and basolateral amygdala and were accompanied by an increase in acetylcholine levels in the brain. The study purported to show early changes due to Gulf War agents, but it did not examine the persistence of these effects.

The hypothesis that stress-induced corticosteroids would prime the CNS to produce a robust proinflammatory response to neurotoxicants and lead to systemic inflammation was tested in male mice. In a 17-day exposure period, the mice were treated with PB/DEET (14 days), corticosterone (7–17 days), and 1 day (day 15) of di-isopropyl fluorophosphate (DFP), a sarin surrogate (O'Callaghan et al., 2015). The level of corticosterone used is known to be immunosuppressive; the weights of the thymus and spleens of all mice receiving it were reduced 20%. DFP caused neuroinflammation throughout the brain in a manner similar to that caused by sarin, with increased expression of multiple proinflammatory mediators. Corticosterone caused a marked exacerbation of the neuroinflammatory effect of DFP. The combination of PB and DEET did not enhance the DFP-induced neuroinflammation with or without corticosterone. There was no effect of DFP, with or without corticosterone, on neurodegeneration, astrogliosis, or microglial activation in the frontal cortex, hippocampus, striatum, or hypothalamus.

Brain Function

PB, permethrin, and DEET exposures combined with restraint stress in rats for 4 weeks reduced numbers of parvalbumin-expressing GABAergic interneurons in some portions of the hippocampus (Megahed et al., 2014). Rats also showed diminished hippocampal neurogenesis at 3 months postexposure. Unfortunately, the ability to apply this information to human health is limited by the use of only 6 rats per group and a relatively high dose of PB (1.3 mg/kg/day). The hippocampus is critical to executive functions, thus this study suggests that, at relatively high doses, PB exposure in the presence of stress could induce delayed neurotoxicity

Other Brain Effects

Williams et al. (2006) used marmosets to study the effect of vaccination or PB on sleep activity and brain electroencephalography. The investigators were blinded as to treatment group. There were no long-term changes in brain electrical activity or sleep architecture that could be attributed to the treatments with vaccines or with PB.

Summary of Brain Effects

Inconsistent results on the permeability of the blood–brain barrier and associated effects to certain combinations of toxic exposures, but not others, give weak support for the hypothesis that stress-induced damage to the blood–brain barrier is a cause for symptoms of Gulf War illness. Three studies showed increased permeability (Abdel-Rahman et al., 2002, 2004; Freidman et al., 1996), but 10 studies found no such effect (Amourette et al., 2009; Grauer et al., 2000; Kant et al., 2001; Lallement et al., 1998; Shaikh and Pope, 2003; Shaikh et al., 2003; Sinton et al., 2000; Song et al., 2002, 2004; Tian et al., 2002). One animal study suggested that stress-induced elevations in cortisone can exacerbate the neuroinflammatory effects of acetylcholinesterase inhibitors (O'Callaghan et al., 2015). Furthermore, altered phospholipid metabolism, which may be associated with adverse effects on the immune system and peroxisomal and lysosomal function, might persist long after exposure to chemicals associated with Gulf War illness (Abdullah et al., 2011, 2012, 2013). Loss of parvalbumin-expressing GABAergic interneurons could be associated with cognitive deficits according to one study (Megahed et al., 2014).

Executive Function

Anxiety and Mood-Related Behaviors

Parihar et al. (2013) examined anxiety-like behavior with a widely used approach—the elevated plus maze. Number of entries into open arms and time spent in open arms was reduced in rats exposed to a combination of PB, DEET, and permethrin, and further reduced when the exposure was combined with restraint stress. These findings are consistent with interpretations of anxiety-like behavior as rats spent more time in the closed areas of the maze.

Several other investigators have measured behavior obtained in an open field, where increased behavior in the periphery of the field compared with behavior in the center of the open field is considered to be an index of anxiety-like behavior. Results have been inconsistent. Hoy et al. (2000b) found increases rather than decreases in time spent by male rats in the central area of the open field in response to PB plus DEET, and to permethrin plus DEET in the immediate 24 hours after a 7-day exposure; a greater time in the center arena was seen in female rats in response to PB plus permethrin following a 7-day exposure. Similarly, mice exposed to PB plus permethrin for 10 days showed a reduction in time spent in the perimeter (i.e., greater time in the center) at day 15 postexposure; however, they spent more time in the perimeter at day 30 postexposure in a study by Abdullah et al. (2011).

Two studies have examined combinations of two or more Gulf War chemicals. Combined exposures of both male and female mice to PB, permethrin, DEET, and restraint stress resulted in more time spent in the perimeter in the 15-minute open field test (Abdullah et al., 2012); however, the differences tend to be about 5 seconds or less, raising questions about their biological significance. Conversely, Hattiangady et al. (2014) examined the effect of the same exposure combination in a similar test in rats and found no effects on time spent in the central zone.

A potentially interesting approach was taken by Servatius and Beck (2005), who examined the possibility that nonspecific symptoms reported in Gulf War illness could reflect classical conditioning effects generated during military service. This hypothesis stipulates that conditioning of symptom effects of toxicant-related exposures could result from environmental stimuli such as sounds or visual cues that would then become conditioned stimuli. Then when these or very similar sounds or visual cues occurred later, the cues could evoke the symptomatology associated with the chemical toxicants. Specifically, the study demonstrated that treatment with PB led to potentiated auditory startle responses occurring in the presence of odors associated with exposures to the stressor (PB), suggesting that such odors could function as conditioned stimuli eliciting PB-related effects. The study found that in rats olfactory stimuli are more significant than visual stimuli; visual stimuli were less or not effective.

Avoidance and escape responses have been examined in two studies. In this paradigm, a cue is presented that signals pending onset of an aversive stimulus (frequently a shock); a designated response after the cue but prior to the onset of the shock is an avoidance response, whereas an escape is defined as a behavioral response occurring in the presence of the shock. Using this approach, Lamproglou et al. (2009) offered rats an opportunity to either avoid or escape shock presentation by climbing a pole. The number of pole climbing trials declined notably across sessions in the PB plus stress groups relative to the stress only group, suggesting more efficient or beneficial avoidance behavior in the former group. This is because climbing the pole prior to the sound initiating the trials actually decreased the numbers of trials (and hence potential shock exposures). No effects of PB, sarin, or the combination on passive avoidance responding were found in rats over a 16-week observation period (Scremin et al., 2003).

Animal models of depression appear limited to a study by Hattiangady et al. (2014) who found rats exposed to a combination of PB, permethrin, and DEET had a reduction in voluntary wheel running and significantly greater latency to eating following a 24-hour fast. Those findings are reminiscent of anhedonia, and as noted by the authors, consistent with decreased motivational levels and depression-like behavior.

As with learning and memory, the studies to date that have examined animal models of anxiety or mood-related disorders do not support any conclusions about the relationship between these disorders and any specific Gulf War-related toxicants.

Motor Function

Most animal studies have evaluated motor function in terms of locomotor activity (ambulation, rearing) and aspects of motor or sensorimotor function.

Summary of Behavioral Effects

The committee finds that it is difficult to arrive at any generalities regarding the behavioral studies discussed in this section for the following reasons:

1.

The studies frequently do not use the same chemicals, doses, or dosing regimens or the same measurement protocols for effects (e.g., timing).

2.

The studies often use different behavioral paradigms to measure the specific behavioral activities.

3.

The relevance of the administered doses to humans is often not evident or the doses are quite high compared with suspected human exposures.

4.

Interpretation of study findings are often difficult given the lack of important data, such as changes in body weights or signs of motivational deficits, which might influence the animal's ability to perform behavioral tests.

5.

The potential for toxicant-induced confounders, including sickness behavior and hypothermia, which also affect behavior, are frequently not reported and not considered in the data interpretation.

Given these issues, it is not possible to draw reliable conclusions about the effects of Gulf War toxicants on animal behavior as measured by learning, memory, mood, or motor function.

Reproductive System

One study assessing sperm and testicular changes in animals exposed to toxicants meant to mirror Gulf War exposures was identified. Male rats were exposed to PB, DEET, and permethrin with and without restraint stress for 28 days (Abou-Donia et al., 2003). Stress alone did not influence these outcomes. Testes of animals exposed to the chemical combination showed histologic abnormalities including arrested spermatogenesis and seminiferous tubule degeneration, effects enhanced under conditions of stress. The authors further investigated the causal mechanism for the observed toxicity and identified apoptosis due to the increased expression of two apoptosis-promoting proteins in testicular tissue. While this study could suggest that exposures such as those experienced by Gulf War veterans may contribute to sperm abnormalities and male infertility, the findings should be viewed with caution, as the animals in the chemical plus stress group also gained less weight, indicating poorer health overall, which compromises the validity of the findings.

Musculoskeletal System

Studies of Gulf War exposures on musculoskeletal function or injury have been carried out in mice and in marmosets. PB and vaccine exposures such as those experienced by Gulf War veterans had no effects on muscle function in marmosets (Stevens et al., 2006). Three 10-week studies in male mice investigated the skeletal muscle effects of combined exposures to PB and exercise stress (Jagannathan et al., 2001; Somani et al., 2000) and PB and/or sarin and exercise stress (Husain and Somani, 2004). Animals were exercised daily, and treatment groups received PB and/or sarin at weeks 5 and 6; doses of PB and sarin were 1.2 mg/kg PB and 0.01 mg/kg sarin (or 1/20th of the LD₅₀). PB with exercise stress, and PB and sarin with exercise stress reduced respiratory exchange ratios during and for several weeks after treatment; the authors suggest that these changes in muscle respiration with and following exercise may enhance oxidative muscle injury. No data was provided on body weight or other indicators of systemic toxicity. The studies in mice suggest a potential mechanism for muscle injury following combined exposures to chemicals and stress designed to mirror those exposures reported by Gulf War veterans.

Immune System

Studies of Gulf War exposures on immune function have been carried out in mice and in marmosets. Little evidence of impaired immune response was observed in marmosets given PB in combination with vaccines (Griffiths et al., 2006; Hornby et al., 2006). Most immune parameters examined were unaffected in female mice exposed to DEET, PB, or JP-8 (jet fuel), singly or in combination (Peden-Adam et al., 2001). All exposures suppressed immunoglobulin-M responses, but this was not exacerbated with mixture exposure. The highest mixture dose increased CD4+ T helper cells, decreased the CD4/CD8 ratio in spleen, and suppressed delayed-type hypersensitivity. In animals treated with melanoma tumor cells and Listeria monocytogenes, susceptibility was not affected (Peden-Adam et al., 2001). As mentioned earlier, lipid metabolism studies in mice exposed to combined PB and permethrin over 10 days suggested an adverse effect on the immune system that lasted long after the exposure (Abdullah et al., 2011).

Pain Response

Nutter et al. (2015) reported a study in which 30 rats were exposed to permethrin, chlorpyrifos, and PB, over 30 or 60 days. The dosages were calculated on the basis of what Gulf War veterans might have experienced and are described as an intensified anticholinesterase exposure protocol relative to earlier studies by this group that did not influence pain behavior (Nutter and Cooper, 2014; Nutter et al., 2013). The exposures did not statistically affect body weight in comparison with controls. Assessment of pain behavior relied on right hind-limb withdrawal in response to force application as well as activity levels (15-minute test periods in an open field). The study reported increases in resting time in the 30-day treated group for approximately 8 weeks postexposure, and the 60-day treated group exhibited increased resting time and reduced movement for 12 weeks postexposure, which the authors interpret as consistent with a delayed myalgia in rats and which was seen in conjunction with a decline in activity of muscle nociceptor channel activity and protein expression.