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

Abstract

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.

Keywords: CGRP; Intranasal delivery; Migraine; Oxidative stress; Reactive oxygen species; Trigeminal ganglion; Trigeminal nucleus.

Figure 1.. Nitroglycerin (NTG)-induced activation in the trigeminal ganglion and trigeminocervical complex.

(A) Trigeminal ganglion oxidative stress (assayed via immunostaining for malondialdehyde) was elevated two hours after intraperitoneal NTG injection compared to vehicle (B) injection. Scale bar (A, B) = 50 μm; representative images from the V1 area of the trigeminal ganglion are shown. Immunostaining results from male animals shown here and throughout were quantified by measuring computer-based fluorescence image intensity. Results were transformed into a ratio of experimental/vehicle intensity which was converted to a natural logarithm and used for statistical analysis (see Methods). (C) NTG versus vehicle injections resulted in a highly significant (***p < 0.001) increase in malondialdehyde immunostaining compared to ND. Similarly, CGRP immunostaining in the trigeminal ganglion V1 region was augmented by NTG (D) compared to vehicle injection (E) that resulted in a highly significant (***p < 0.001) Ln ratio compared to no difference (ND) (F). Scale bar (C, D) = 50 μm; representative images are shown. Finally, representative images (for illustrative purposes shown at −6.0 mm from the obex, that are similar to positive staining found at all other levels), show NTG (G) or vehicle (H) c-Fos positive immunostaining in the trigeminocervical complex from a male animal. Scale bar (G, H) = 250 pm. (I) Statistical treatment of Ln ratios of experimental/vehicle cumulative (left plus right from sections at 0, −1.5, −3.0, −4.5 and −6.0 mm from the obex) c-Fos positive cells show that NTG triggered a significant (***p < 0.001) increase in c-Fos labelled trigeminocervical complex positive cells compared to vehicle.

Figure 2.. Intranasal delivery of IGF-1 significantly reduced NTG-induced activation in the trigeminal ganglion and trigeminocervical complex.

Nasal pretreatment with IGF-1 (A) reduced the NTG-induced increase in malondialdehyde immunostaining in the V1 area of the trigeminal ganglion compared to nasal pretreatment with succinate vehicle (B). Scale bar (A, B) = 50 μm. (C) Statistical analysis showed that nasal IGF-1 significantly (***p < 0.001) reduced the Ln ratio (IGF-1+NTG/succinate+NTG) impact on fluorescence image intensity compared to no difference (ND). Similarly, nasal pretreatment with IGF-1 attenuated the NTG elevation in CGRP trigeminal ganglion V1 area immunostaining (D) compared to nasal succinate vehicle pretreatment (E) and showed a highly significant (***p < 0.001) Ln ratio difference compared to no difference (ND) (F). Finally, the NTG induced increase in c-Fos positive cells in the trigeminocervical complex was also reduced by nasal pretreatment with IGF-1 (G) compared to nasal succinate vehicle pretreatment (H). Representative sections shown here at −6.0 mm from the obex. Scale bar (G, H) = 250 μm. In all cases, representative images from low dose (37.5 μg) IGF-1 versus succinate vehicle treatment in males are shown. Statistical treatment of Ln ratios of experimental/vehicle cumulative (left plus right from sections at 0, −1.5, −3.0, −4.5 and −6.0 mm from the obex) c-Fos positive cells show that nasal IGF-1 triggered a significant (***p < 0.001) reduction in c-Fos labelled trigeminocervical complex positive cells compared to vehicle.