Insulin-like growth factor-1 inhibits nitroglycerin-induced trigeminal activation of oxidative stress, calcitonin gene-related peptide and c-Fos expression

Migraineurs experience increased oxidative stress which drives the initiation and maintenance of migraine-related pain in animal models and, by extension, migraine in humans. Oxidative stress augments calcitonin gene-related peptide (CGRP) levels, a mediator of migraine pain. Insulin-like growth factor-1 (IGF-1), a neuroprotective growth factor, reduces susceptibility to spreading depression, a preclinical model of migraine, in cultured brain slices by blocking oxidative stress and neuroinflammation from microglia. Similarly, nasal delivery of IGF-1 inhibits spreading depression in vivo. After recurrent cortical spreading depression, nasal administration of IGF-1 also significantly reduces trigeminal ganglion oxidative stress and CGRP levels as well as trigeminocervical c-Fos activation. Here, we probed for the impact of nasal IGF-1 pretreatment on trigeminal system activation using a second well-established preclinical model of migraine, systemic nitroglycerin injection. Adult male rats were treated with one of three doses of IGF-1 (37.5, 75 or 150 μg) and the optimal dose found in males was subsequently used for treatment of female rats. One day later, animals received an intraperitoneal injection of nitroglycerin. Measurements taken two hours later after nitroglycerin alone showed increased surrogate markers of trigeminal activation - oxidative stress and CGRP in the trigeminal ganglion and c-Fos in the trigeminocervical complex compared to vehicle control. These effects were significantly reduced at all doses of IGF-1 for trigeminal ganglion metrics of oxidative stress and CGRP and only at the lowest dose in both males and females for c-Fos. The latter inverted U-shaped or hormetic response is seen in enzyme-targeting drugs. While the specific mechanisms remain to be explored, our data here supports the ability of IGF-1 to preserve mitochondrial and antioxidant pathway homeostasis as means to prevent nociceptive activation in the trigeminal system produced by an experimental migraine model.


Introduction
Migraine is a common neurological disorder with a female-to-male ratio of 3:1 and a yearly prevalence of approximately 15 % [1]. Migraine ranks second among all diseases associated with disability [1]. In the United States alone, migraine results in annual costs of $78 billion [2]. As a result, migraine is an immense heath care and societal burden.
A large subset of migraineurs experience recurrent [i.e., episodic (0-14 headache days/month) or chronic (15 or more headache days/ month)] migraines [3] which often suggest that the use of daily medications may be of benefit [4]. Approved therapies for recurrent migraine such as topiramate, amitriptyline or beta blockers offer only fractional protection and include significant adverse effects that substantially reduce their use [5]. Similarly, botulinum toxin injections, FDA approved only for chronic migraine, have a significant albeit modest reduction in migraine frequency of only two days/month [6]. Calcitonin gene related peptide (CGRP) is an inflammatory molecule involved in migraine pain [7]. Newly developed anti-CGRP biologics for treatment of recurrent migraine are antibodies that inhibit or absorb circulating CGRP to block nociceptive activation of the trigeminal system [8]. While promising, clinical efficacy in recurrent migraine patients may be limited [9]. About 30-50 % of the patients treated via injections/infusions with these new drugs responded and saw their migraine days reduced by 50 % from 8 to 10 to 4-6 migraines per month, or around 20 % over placebo [4]. Collectively, this speaks to the continued need for development of new therapeutics for treatment of migraine.
Preclinical models are important for the development of migraine therapeutics. Cortical spreading depression (CSD), the likely cause of migraine aura and perhaps migraine related head pain, is a wellrecognized model of migraine [10]. Ayata and coworkers show that traditional migraine therapeutics inhibit CSD, supporting use of CSD as means to screen for new migraine therapeutics [11]. This model was applied to study the impact of insulin-like-growth factor-1 (IGF-1) on CSD susceptibility, since IGF-1 is known to protect against ischemic injury [12] and promote neural health [13]. Studies show that IGF-1 significantly reduces susceptibility to CSD in vitro using cultured brain slices [14] and in vivo after nose-to-brain (i.e., referred to here and throughout as "intranasal") administration in rats [15]. Nasal delivery of IGF-1 also significantly inhibits head pain-related trigeminal system activation from CSD. This latter effect involves reductions in trigeminal ganglion oxidative stress as well as CGRP and trigeminocervical c-Fos activation [16].
Systemic injection of nitroglycerin (NTG) is a second wellestablished preclinical model of migraine [10,17]. Like, CSD, NTG administration has recently been validated as an important translational model for screening migraine therapeutics. Akerman and coworkers show that newly approved clinical treatments for migraine, gepants and ditans, effectively reduce cranial hypersensitivity, a metric for nociceptive head pain activation after NTG [18]. Here, we probed for the impact of nasal IGF-1 after systemic NTG administration. Results showed that as with CSD, nasal delivery of IGF-1 significantly reduced trigeminal ganglion oxidative stress and CGRP levels and reduced trigeminocervical c-Fos activation in the NTG model.

Animals
Adult male (n = 40, 260− 410 grams) and female (n = 10, 204− 250 grams) Wistar rats (Charles River Laboratories, Wilmington, MA) were used in this study and housed two/cage until treatments, after which they were housed one animal per cage as previously described [16].
All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Chicago, were conducted in accordance with the Guidelines of the National Institutes of Health Guide for Care and Use of Laboratory Animals (2011) and were patterned after ARRIVE guidelines. In addition, all experiments were performed in accordance with EC Directive 86/609/EEC for animal experiments and Uniform Requirements for manuscripts submitted to Biomedical journals recommended by the International Committee of Medical Journal Editors. Unless otherwise indicated, all routine chemicals were purchased from Sigma or ThermoFisher.
Experiments were performed during the mid-portion of the light cycle. Rats were randomly divided into control and experimental groups. Sample sizes (n = 5/group) were based on our previous published studies that show a significant change and Power of always greater than 0.8 and most often equal to 1.00. All treatments and data analyses were completed under blinded conditions.

Drug treatment
NTG was obtained at a stock concentration of 5.0 mg/mL in 30 % alcohol, 30 % propylene glycol, and water (American Regent Inc., Shirley, NY), diluted with sterile saline (0.9 % NaCl) to 1.0 mg/mL and administered by intraperitoneal (i.p.) injection at a dose of 10 mg/kg [17,19,20]. The NTG vehicle used for these experiments consisted of 6% alcohol, 6% propylene glycol in 0.9 % NaCl instead of saline to negate the potential confound between group comparisons that otherwise might occur from vehicle constituents [21].
24 h prior to NTG injection, rats received intranasal treatment with sodium succinate buffer, or 37.5, 75 or 150 μg human recombinant IGF-1 [(IGF-1), #191-G1; R&D Systems, Minneapolis, MN] in 50 μl sodium succinate buffer following our previously described procedures under inhalational isoflurane anesthesia with monitoring [15,16]. No adverse effects (i.e., altered feeding, grooming or ambulatory behavior) were seen from intranasal treatments in this work or our prior experiments that involved treatment with IGF-1 for up to two weeks [15]. Rats were randomly allocated to experimental groups as follows. In a first experiment groups of males were given: 1) intranasal succinate buffer/intraperitoneal NTG or 2) intranasal succinate buffer/intraperitoneal NTG vehicle; (n = 5/group). In a second set of experiments groups of males were given: 3) 37.5 μg intranasal IGF-1/intraperitoneal NTG; 4) 75 μg intranasal IGF-1/intraperitoneal NTG; or 5) 150 μg intranasal IGF-1/intraperitoneal NTG. For the second set of experiments each IGF-1treated group had a corresponding control group [intranasal succinate/intraperitoneal NTG (n = 5/group)]. In a third experiment, females were treated at the optimal IGF-1 dose found in experiment two. Each animal was used for trigeminal ganglion and trigeminocervical complex measurements.

Immunostaining
Two hours following intraperitoneal injection of NTG [22], animals were anesthetized by intraperitoneal injection of ketamine/xylazine, perfusion-fixed and neural tissue harvested as previously described [16].
Trigeminal ganglia were cut into 20 μm thick, consecutive, longitudinal sections using a cryostat (#3050S; Leica, Buffalo Grove, IL) and mounted as one section per gelatin coated slide. Sections containing the V1 (ophthalmic) division of the trigeminal ganglion were identified and three sections, at least 40 μm apart, from each animal were processed for malondialdehyde, a marker of oxidative stress [23], and CGRP immunostaining as previously described [16].
For c-Fos immunostaining of the trigeminocervical complex, the brainstem/cervical spinal cord was cut into 40 μm thick coronal sections using a cryostat. Laterality was controlled for by marking the right ventral aspect of the brainstem/cervical spinal cord. Six consecutive sections were collected every 1.5 mm from the obex (0 mm) caudally through cervical spinal cord (i.e., -1.5 mm, -3.0 mm, -4.5 mm, -6.0 mm) as previously described [16,24]. For each animal, one section was randomly selected from each brainstem/spinal cord level and processed for c-Fos expression. c-Fos immunohistochemistry was performed on free floating sections and endogenous peroxidases eliminated by incubating in 1% hydrogen peroxide in PBS for 15 min prior to blocking with 5% normal goat serum containing 0.3 % Triton X-100 for one hour at room temperature. The sections incubated overnight in rabbit anti-c-Fos (1/10,000, ABE457, Millipore, Temecula, CA) in 1% normal goat serum/0.3 % Triton X-100 at 4 • C and then in biotinylated goat anti-rabbit secondary antibody (1/200, BA-1000, Vector Laboratories, Burlingame, CA) for 2 h. Afterward, sections were processed using the Vectastain Elite ABC kit (PK-6100, Vector Laboratories). c-Fos staining was visualized using 3 ′ ,3 ′ -diaminobenzidine tetrahydrochloride and sections mounted on gelatin coated slides then coverslipped with Permount. Specificity of the immunolabeling was confirmed by omitting the primary antibody, using only secondary antibody staining.

Computer-based digital image quantification
Computer-based and blinded semi-quantitative digital quantification of immunostaining metrics was used to test the ability of intranasal IGF-1 to impact trigeminocervical complex activation as previously described [16]. To reduce inter-experiment variability, all immunostaining was performed on paired samples (e.g., sham and experimental) sections for all markers (malondialdehyde, CGRP and c-Fos).
For malondialdehyde and CGRP, imaging was performed using digital imaging strategies as previously described [16]. Resultant images were digitally stored as TIFF files for subsequent analyses that continued under blinded conditions where image integrated fluorescence intensity was registered using MetaMorph software. The average fluorescence image intensity of three technical replicates was registered for statistical analyses.
For c-Fos image analyses, 5x bright field images were photographed (Leica LMD6000) and stored as TIFF files. Photoshop CC® (Adobe®, San Jose, CA) was used to convert images to 1200 dpi and set a uniform brightness and contrast as well as to draw borders to delineate areas of the trigeminocervical complex [i.e., laminae I-V of trigeminal nucleus caudalis and C1/C2 cervical spinal cord dorsal horn as described by Strassman and coworkers [25] for quantitating c-Fos labelled nuclei. Induction of c-Fos expression has been reported to occur in these anatomical regions two hours following intraperitoneal NTG administration in mice [22]. c-Fos labeled nuclei were manually counted bilaterally in paired samples (i.e., experimental and sham) by an observer blinded to experimental conditions. Manual cell counts were tabulated using the cell counter function in ImageJ. Only intensely stained, round or oval-shaped nuclei were counted [-1.5 mm through -6.0 mm from the obex], and the sum of c-Fos labeled nuclei of both left and right sides at all levels from a representative section at each anatomical level per animal was recorded.

Statistical methods
We used a verified method of quantifying immunostaining log-ratios (experimental / sham) as a further means to reduce potential run-to-run variations in immunostaining for malondialdehyde, CGRP and c-Fos positive cells [16].
Images of malondialdehyde and CGRP were analyzed in blinded pairs so that they could be converted once decoded to ratios of experimental/sham image integrated optical intensity, a sensitive metric that not only accounts for the area but also the pixel intensity of related image fluorescence. Immunostaining ratios of one indicated no difference between experimental and sham conditions, while a ratio of greater than one indicates a change greater than sham and a ratio less than one indicates that experimental treatment reduced immunostaining compared to sham. These ratios were converted to natural logarithms whereby "0" corresponds to no difference between experimental and sham conditions, and a t-test (two-tailed) could be used to determine if differences of logarithms varied significantly from 0. In Tables 1 and 2 we report p-values and power of related log ratio statistical testing. In addition, a more general listing of percent changes in intensity ratios is listed in the text.
c-Fos positive cell quantifications followed a similar pattern to that used for malondialdehyde and CGRP immunostaining. Specifically, total c-Fos positive cell counts were converted to a ratio (i.e., IGF-1/sham) and the natural logarithm of the results quantified statistically by comparison to "zero" (i.e., no difference ratio of 1.00 or Ln = 0).
All image pairs (experimental and sham) were adjusted equally using Photoshop®, which along with CorelDraw x7 (Ottawa, Canada) was used for final figure construction. Data were analyzed using SigmaPlot software (v. 12.5; Systat Software, Inc. San Jose, CA). All data passed normality testing (p-value to reject: 0.05) and equal variance testing (pvalue to reject: 0.05) and power (1-β: > 0.8). Animal groups consisted of five biological replicates.

Results
We first confirmed that intraperitoneal injection of NTG in Wistar rats significantly elevated trigeminal system metrics of nociceptive activation (i.e., trigeminal ganglion oxidative stress measured as malondialdehyde and CGRP protein levels as well as trigeminocervical complex c-Fos activation) [26,27]. In each case, two hours after intraperitoneal NTG, nociceptive metrics were significantly elevated, consistent with the time point for activation found by others for both intraperitoneal NTG in mice [22] and intravenous NTG in rats [28].
Relative ratios (i.e., intranasal succinate + intraperitoneal NTG / intranasal succinate + intraperitoneal NTG vehicle) showed a 325 % increase in oxidative stress (malondialdehyde) and 332 % increase in CGRP in the trigeminal ganglion as well as 183 % increase in c-Fos positive cells from the summed left and right trigeminocervical complex sections after NTG. Representative immunostaining images and Ln ratio changes are shown in Fig. 1. Specific Ln ratios and statistical comparisons to no difference (i.e., Ln = 0) are listed in Table 1. In each instance, these nociceptive metrics were significantly increased after intraperitoneal NTG.
Previous work shows that systemic therapeutic administration of IGF-1 (e.g., for treatment of short stature due to primary IGF-1 deficiency in children) can be associated with transient hypoglycemia and headache [29]. However, intranasal (i.e., direct nose-to-brain) delivery of IGF-1 in rats shows no evidence of causing hypoglycemia [16]. Furthermore trigeminal ganglion CGRP, a measure of potential headache pain is actually reduced in naïve animals after intranasal IGF-1 treatment, suggesting that this route of delivery would not induce headache and could be antinociceptive [16].
In a second series of experiments, we probed for the ability of nasal treatment with IGF-1 to mitigate trigeminal system nociceptive activation two hours after intraperitoneal NTG. Dose-response studies were performed in male rats and the optimal dose from these results was used for nasal IGF-1 pretreatment in female rats (Supplemental Fig. 1). Relative ratio (i.e., IGF-1+NTG/succinate + NTG) results showed that high (150 μg), medium (75 μg) and low (37.5 μg) doses of intranasal IGF-1 were highly effective in reducing NTG-induced trigeminal ganglion oxidative stress (malondialdehyde) in male rats by 77 %, 49 % and 65 %, respectively. Similarly, high, medium and low intranasal doses of IGF-1 were also highly effective in reducing relative CGRP ratios by 67 %, 68 %, and 56 %, respectively. Also, treatment with the low dose of  intranasal IGF-1 (chosen for impact on c-Fos; see below) in female rats produced a 63 % reduction in malondialdehyde-related oxidative stress and a 74 % reduction in CGRP levels. Specific Ln ratios (IGF-1+NTG/ succinate + NTG) and statistical comparisons to no difference (i.e., Ln = 0) are listed in Table 2. Representative immunostaining images and Ln ratio changes for males are shown in Fig. 2 (and females in Supplemental Fig. 1). NTG-induced increase in trigeminocervical complex c-Fos expression was not significantly reduced by treatment with nasal IGF-1 at the high (150 μg) or medium (75 μg) dose. In contrast, low (37.5 μg) dose nasal IGF-1 triggered a 22 % reduction in c-Fos positive cell labeling in males and a 44 % reduction in females. In both instances these changes were highly significant (Table 2) with male representative results shown in Fig. 2.

Discussion
This study shows that as seen after recurrent CSD [16], pretreatment with nasal IGF-1 is an effective means to inhibit trigeminal system activation after intraperitoneal NTG, a second well-accepted model of migraine [26]. The successful impact of nasal IGF-1 on mitigating trigeminal system nociceptive metrics in a second model of migraine provides important additional evidence supporting further development of this growth factor as a novel migraine preventative therapeutic.
Delineation of mechanisms by which nasal IGF-1 protects against trigeminal nociceptive activation after NTG is beyond the scope of this work. However, current literature and the pattern of response reported here can begin to suggest potential signaling systems involved. Extensive evidence indicates that NTG leads to trigeminal nociceptive system activation. Li and coworkers [38] show significant elevation of brain tissue oxidative stress markers following NTG administration. Additionally, trigeminal ganglion levels of hydrogen peroxide rise two hours after intraperitoneal injection of NTG, and after four hours 4-hydroxynonenal, a product of lipid peroxidation in cells, is significantly increased in the ganglion [27].
While multiple mechanisms have been suggested to explain NTGinitiated trigeminal system activation and headache, the work of Marone and coworkers [27] seems particularly important to begin understanding the effects of nasal IGF-1 on trigeminal system activation reported here. The conversion of NTG to nitric oxide stimulates oxidative stress [31] that directly targets TRPA1 channels, well-recognized sensors for pain signaling, including that potentially from migraine [32]. TRPA1 channel activation in trigeminal ganglion neurons in turn prompts prolonged production of reactive oxygen species (ROS), which can further activate trigeminal ganglion neurons in a positive feedback loop prompting increased release of CGRP. Furthermore, periorbital allodynia, a delayed effect of NTG, can be reversed by treatment with ROS scavengers after NTG administration [27], emphasizing the role of oxidative stress and ROS in migraine models and potentially migraine patients. Additionally, most existing prophylactic migraine agents show some degree of antioxidant effect [33] providing further support for targeting oxidative stress in migraine treatment. Depletion of antioxidants seen in migraine leads to worsening of this disorder [34]. Our results show that nasal IGF-1 significantly reduces NTG-induced trigeminal ganglion oxidative stress by 49-77 % (measured by malondialdehyde immunostaining) over a range of doses in male rats as well as in females by 63 % at the lowest dose of 37.5 μg used in males). Given that females are generally more sensitive to migraine activation, it is notable that our optimal IGF-1 dose from males was nonetheless significantly effective in all nociceptive metrics in females. Future studies will explore this in detail.
Increased trigeminal ganglion oxidative stress leads to significantly elevated trigeminal ganglion CGRP levels [27,35], which are closely involved in migraine pain pathophysiology [8]. Accordingly, CGRP immunostaining was used here as a second metric for trigeminal ganglion nociceptive activation potentially associated with headache. Neuropeptides such as CGRP are synthesized in cell bodies and move to the axon terminal for release [36]. For subsequent new release, more peptides must be produced in neuronal somata. Thus, cell soma levels of CGRP can be expected to reflect the CGRP that can be released by trigeminal ganglion activation. If so, then the IGF-1-related changes in trigeminal ganglion oxidative stress and CGRP expression support a potential pain relieving ability of IGF-1 in migraine. In this study, nasal IGF-1 effectively prevented the NTG-induced increase in trigeminal ganglion CGRP levels by 50-77 % in males over a range of doses, as well as in females by 74 % at the lowest dose of 37.5 μg [e.g., most effective dose for c-Fos change seen in males (see below)]. Notably no evidence of a dose-dependent impact of nasal IGF-1 was seen in trigeminal ganglion activation metrics of oxidative stress and CGRP.
Trigeminal ganglion activation leads to related trigeminocervical activation, evidenced here and by others by increased expression of c-Fos. Bates and coworkers [22] show that two hours after intraperitoneal injection of NTG, c-Fos is significantly elevated in laminae I-V of the trigeminocervical complex, a measurement region applied here. Others too show that NTG administration consistently results in enhanced trigeminovascular c-Fos expression [22,28,30,37]. Consistent with the increased incidence of migraine in females compared to males, Greco and colleagues show greater expression of c-Fos in female compared to male rats four hours following intraperitoneal NTG administration [37]. Also, female mice develop basal mechanical hyperalgesia more quickly than male mice after chronic intraperitoneal NTG [38] and the magnitude of mechanical hyperalgesia is greater in female compared to male rats after acute intradermal administration of glycerotrinitrate [39]. Despite sex related differences in behavioral response, anti-migraine agents, sumatriptan and topiramate, have been shown to effectively attenuate NTG induced mechanical hyperalgesia in both female and male mice [38]. Importantly, our results show that nasal treatment with IGF-1 significantly reduces trigeminocervical c-Fos activation after NTG administration in male as well as female rats. Results show that a significant reduction (22 %, compared to sham) in c-Fos immunostained cells was seen at the lowest dose of (37.5 μg of IGF-1) but not at higher doses in male rats. When administered to female rats, nasal IGF-1 also produced a significant (44 %) reduction in c-Fos positive cells compared to sham. This inverted or U-shaped dose response of IGF-1 on c-Fos activation in the NTG migraine model is not unusual (see below). However, it is in contrast to dose-response effects seen with nasal IGF-1 pretreatment (37.5, 75 and 150 μg) in a CSD model of migraine which showed a linear response with significant changes for all trigeminal ganglion metrics measured here as well as a significant (45 %) reduction in c-Fos positive cells in the superficial layers of the trigeminocervical complex at the 150 μg dose of IGF-1 (See Supplemental Table 1). Potential bases for these results from IGF-1 are discussed below.
While trigeminal ganglion and the trigeminocervical complex are synaptically connected and so their nociceptive activation might be expected to occur in tandem. However, this simple expected direct relationship may be complicated with anti-nociceptive treatments. Our data points to this suggestion.
Nitric oxide leads to trigeminovascular activation of cellular c-Fos via two routes. The first is through increased synaptic activity from trigeminal neurons to trigeminocervical complex nociceptive neurons, evidenced by the surrogate markers for increased neuronal activity of oxidative stress and CGRP. The second can involve a direct effect of nitric oxide on second order nociceptive neurons in the trigeminocervical complex. In both instances, nitric oxide activates c-Fos by a series of enzymatic steps (leading from nitric oxide to activation of soluble guanyl cyclase, cyclic guanosine monophosphate, protein kinase G, phosphorylated cAMP response element-binding protein and finally c-Fos) [19,40]. In addition, NTG mediated neuronal activation in the trigeminocervical complex can result from increased glutamatergic tone. Kynurenic acid is an endogenous glutamate receptor antagonist produced by the kynurenine pathway. Enzymes related to kynurenine synthesis and metabolism are downregulated by NTG, suggesting a potential added impact on neuronal activation from increased glutamate acting upon post-synaptic N-methyl-D-aspartate receptors [41]. In contrast to NTG, CSD increases brain kynurenic acid levels [42]. Thus, although both NTG and CSD activate the trigeminovascular system, they employ different physiological mechanisms. Contrary to what we expected, the lowest dose of IGF-1 (37.5 μg) was effective in reducing NTG induced c-Fos in the trigeminocervical complex. Reasons for this are unclear, but could involve hormetic or U-shaped dose-response mechanisms which are well-recognized to occur in biological systems including those seen from the administration of enzyme-targeting drugs [43]. Alternatively, it is possible that that IGF-1 can simply evoke a hormetic response involving low dose stimulation and high dose inhibition as described for IGF-1 effects on potassium channel activity [44]. Such a biphasic mechanism of IGF-1 on neuronal activation (c-Fos) in the NTG migraine model would need to involve NTG-related signaling pathways distinct from CSD that more likely relies upon synaptic activity. Future studies will help resolve differences in cellular signaling between CSD and NTG-induced trigeminocervical c-Fos activation. However, the key element to the current study, and previous work involving IGF-1 and recurrent CSD, is that nasal IGF-1, at all doses in males and the optimal dose from males used in females, significantly reduced trigeminal ganglion activation, a necessary concomitant of migraine modeled in animals and likely that seen in humans suffering from this malady.
IGF-1 reduces oxidative stress. IGF-1 treatment of hippocampal brain slices in vitro, reduces susceptibility to spreading depression by inhibiting the generation of ROS and pro-inflammatory cytokines from microglia that otherwise promote spreading depression [14]. Recurrent CSD leads to increased malondialdehyde a product of lipid peroxidation from increased ROS in trigeminal ganglion cells [23], an effect that is reversed by pretreatment with nasal IGF-1 [16] and also shown to occur here after NTG treatment. Neuronal activation generates increased production of ROS at least in part due to the comparatively high number of mitochondria found in these cells [45]. IGF-1 is known to reduce ROS produced by exposure of hippocampal brain slices in vitro to menadione, a mitochondrial inhibitor [14]. Evidence shows that IGF-1 protects mitochondrial function and so reduces ROS production via Nrf2 pathway signaling [46] which may involve enhanced antioxidant production via upregulation of glutathione pathway signaling [47].
In conclusion, nasal administration of IGF-1 effectively mitigates the trigeminal system (ganglion and trigeminocervical complex) nociceptive activation that otherwise would be seen hours after systemic NTG treatment, results that parallel findings seen previously with CSD [16]. These effects likely involve the potent antioxidant effects of IGF-1 on mitochondria and antioxidant pathways. Notably, reduced antioxidant status is seen in migraineurs [34] as well as mitochondrial dysfunction [48], suggesting the potential utility of nasal IGF-1 administration to attenuate oxidative stress triggers contributing to migraine.
Finally, migraine pain-related behavior is strongly correlated with trigeminal system CGRP upregulation, one of the metrics that was significantly reduced by intranasal IGF-1 [49,50]. However, to bolster this correlation, future studies will need to explore the impact of IGF-1 on mitigating pain-related behavior from NTG as well as its potential mechanisms. The magnitude of IGF-1-related reductions in trigeminal ganglion oxidative stress and CGRP may be related to the fact that the trigeminal ganglion attains the highest brain levels of IGF-1 after nasal delivery [51].

CRediT authorship contribution statement
Drs. Lisa Won and Richard Kraig: participated equally in the design, execution and interpretation of all data as well as writing the final manuscript.

Disclosures
Richard P. Kraig is a co-inventor (along with others) on issued and pending patent applications dealing with "Treatments for migraine and related disorders".
Richard P. Kraig is also a co-founder and the Chief Scientific Officer of Seurat Therapeutics, Inc., a company formed to develop IGF-1 as a novel treatment for migraine.
Dr. Lisa Won has no conflict. All aspects of this study passed review by the Provost's Office of the University of Chicago which included assurances on an external oversight monitor for the work and blinding of all experiments.
Drs. Lisa Won and Richard Kraig participated equally in the design, execution and interpretation of all data as well as writing the final manuscript.