We kindly thank Luiz Henrique Moreira de Souza for the supporting illustrations.

All procedures were approved by the ethical guidelines of the International Association for the Study of Pain regulations on animal welfare and the National Institutes of Health guidelines for the care and use of laboratory animals. The Institutional Animal Care and Use Committee of the University of Arizona previously approved the experimental procedures described in this protocol.
NOTE: The mTBI model described in this protocol is optimized for female or male C57/BL6J12</su...

Herein, we describe a mouse model of mTBI in which the head is unfixed, reproducing many biomechanical aspects of common human mild head injuries, including closed-skull, blunt force direct impact, and linear, rotational, acceleration, and shearing forces from unrestrained head movement. It is suggested that these similarities of the model to human concussion might allow for increased relevance of investigations of pathophysiological mechanisms associated with APTH and the transition to PPTH. The injury produced by this ...

Mild traumatic brain injury (mTBI), also known as concussion, represents the most prevalent type of closed head injury and is one of the leading causes of global disability1,2,3,4,5. mTBI often affects civilians, military personnel, and veterans and can result from blunt trauma, acceleration, or deceleration forces, including falls, motor vehicle collisions, contact or collision sports, exposure to blasts, and additional mechanisms of injury1,2. Post-traumatic headache (PTH) is the most common and often incapacitating symptom following mTBI. Headache attributed to mTBI that resolves within 3 months is considered as acute PTH (APTH), while headache symptoms that persist beyond 3 months and can last for years are defined as persistent PTH (PPTH)1,2,3. Approximately 50% of individuals who develop APTH after mTBI will progress to PPTH6.
In spite of significant advances in clinical studies of PTH, there is still no approved therapy for this condition1,2,7. The pathophysiological changes underlying PTH and/or those driving transition to PPTH remain poorly understood. Multiple mechanisms have been suggested to contribute to PTH/PPTH, including genetic phenotype, prior existing conditions such as migraine, neuroplasticity of descending pain modulatory pathways, brain structural and functional remodeling, and others8. PTH most often presents with a migraine phenotype, suggesting overlapping pathophysiological mechanisms, including the likely involvement of calcitonin-gene-related peptide (CGRP) signaling1,7. Nevertheless, clinical reports suggest that most migraine therapeutics, including those modulating CGRP are not very effective for PTH9. Further studies are warranted for a better understanding of pathophysiological mechanisms associated with PTH in order to discover improved therapies to control PTH and to prevent the progression to PPTH.
Animal models of mTBI ideally should closely consider clinical symptoms in order to increase potential mechanistic translation. Recently, models of TBI have been classified into three main categories based on the method of induction. These methods include weight drop, piston-driven impacts, and &#34;other&#34; models that are described as &#34;unconventional&#34;10. The weight drop models constitute the largest group and consist of dropping a projectile with specific characteristics (material, size, weight) through a guide tube placed at a specific height directly onto the head of the animal10. Extensive variations of the weight drop method to induce mTBI in rodents can be found in the literature10. Piston-driven models of mTBI, such as controlled cortical impact (CCI), consist of a piston that produces injury related to a specific depth, velocity, and/or impact force when considering the surface of the skull or scalp as the zero or the hit mark10. Impact devices can include pistons driven by compressed nitrogen, electromagnetic or pneumatic mechanisms10. In addition, penetrating injuries may also be induced by a pulse of pressurized fluid (i.e., fluid percussion, FPI) through a craniotomy7. However, while the exact method and the severity of injury induced by CCI and FPI can be varied, they often produce more severe injuries with substantial brain tissue loss and neuropathological outcomes that may better resemble severe TBI versus the mild TBI concussive injuries most commonly associated with PTH and PPTH in humans7. Most of the studies using the weight drop and the piston model of mTBI have used adult mice and rats with additional variables that included adaptations according to the species, strain, and size of the rodent10. Many other types of mTBI models have also been described in the literature, including a spring-loaded launching, the projectile model, a rubber reflex hammer fixed into a pivot, a microTBI surgical technique, and a model of fall, which induces mTBI by dropping the animals onto an immovable surface10.
Our group has adapted a weight drop model of mTBI allowing free rotational and linear head movements, previously described by Khun and colleagues11,12,13,14. The weight drop apparatus used to mimic mTBI induction reported in this protocol is represented in Figure 1. It consists of a vertical guide metal tube (124 cm) that holds and guides the weight that is dropped onto the head of a lightly anesthetized mouse. A PMMA (Poly(methyl methacrylate)) box (20 cm x 20 cm x 16.5 cm) with an opening on the top (12 cm x 8 cm) allows the placement of the animal and allows the linear and rotational movement of the head after the impact from the weight drop. The bottom of the box contains one 2.5 cm-thick wavy and two 1.25 cm-thick flat acoustic foam cushions (15 cm x 18 cm) to support and comfort the animal after the impact from the weight drop. The cushion and the bottom of the PMMA box also have an opening through which the weight goes, preventing a second hit during the fall of the animal. The opening of the top of the PMMA box is covered by a single layer of tissue paper, with perforation (11 cm x 12 cm) with the ends taped, which supports the body weight of a mouse up to 50 g for mTBI impact or sham procedures (no impact). The thinness of the paper stage produces little or no resistance upon the weight drop impact. The projectile to induce the weight drop (20 cm and 0.3 cm diameter) is made of steel, weighs 100 g, and has a flat end with radiused edges (0.3 cm) to restrict the zone of contact to the top of the head between the ears and the eyes of the mouse. The weight is released from a height of 37 inches above the head. The weight drop impacts the sagittal suture midway between bregma and lambda. The weight is placed on the top of the guide tube, with the flat end with radiused edges pointing down toward the animal, and is held in place by an L-shaped steel trigger (5 cm x 2 cm). The weight is dropped vertically through the guide tube by removing the trigger.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63444/63444fig01.jpg" /> 
Figure 1: The weight drop apparatus for mTBI induction to the unfixed head of a mouse. (A) The apparatus consists of a vertical guide metal tube (124 cm) for the weight drop and a PMMA box (20 cm x 20 cm x 16.5 cm) with a 5 cm acoustic foam cushion in the bottom (one 2.5 cm thick wavy acoustic foam and two 1.25 cm thick flat acoustic foam) (B). The 100 g steel weight with (C) dimensions (20 cm and 0.3 cm diameter) has a flat end with radiused edges (0.3 cm), which is placed on the top of the guide tube and is held in place by a steel trigger (red circle on panel A). The weight is released from a height of 94 cm above the head. (D) The top of the PMMA box has a 12 cm x 8 cm opening to allow the weight to pass through, applying both linear and rotational acceleration during the impact. The acoustic foam and the bottom of the PMMA box also have a 0.6 cm x 1.2 cm opening to allow the passage of the weight drop and avoid re-hits. <a href="https://www.jove.com/files/ftp_upload/63444/63444fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
This model effectively reproduces many of the biomechanics associated with the injury, including an impact to an unrestrained head, allowing the consequences of both linear forces and rotational acceleration. Using this model in mice, we described the presence of a period of initial transient cutaneous allodynia (CA) lasting several days after mTBI induction revealed by increased frequency of response to tactile stimulation of the periorbital (i.e., cephalic) and hind paw (i.e., extra-cephalic) regions14, suggestive of central sensitization. CA typically persists until day 7 after the mTBI and returns to baseline thresholds by approximately day 14. People with primary headaches often show disruption of central pain modulatory pathways measured by the loss of conditioned pain modulation (CPM)15,16,17,18. Disruption of central pain modulatory pathways has also been observed in individuals with PTH in early and later stages after mTBI19,20,21,22. Correspondingly, we have demonstrated disruption of central pain modulatory pathways following mTBI in this model by the loss of descending noxious inhibitory controls (DNIC), a preclinical measure of CPM in humans13. Loss of DNIC was observed in the first few days after mTBI induction13. Time-dependent allodynia and loss of DNIC following mTBI represent pain-like behaviors in the APTH.
People with migraine and PTH commonly report stress or its relief as a trigger that initiates or exacerbates their attacks21,23,24,26. For this reason, we examined whether stress would elicit allodynia in mice that have recovered from the APTH. Mice were exposed to psychological stress induced by exposure without restraint to bright lights (i.e., bright light stress, BLS) on day 14 following resolution of initial mTBI-induced CA. The BLS reinstated periorbital and hind paw CA only in mice previously subjected to the mTBI14. The model allows adaptation of the &#39;&#39;two-hit&#39;&#39; strategy of hyperalgesic priming27, wherein the initial noxious stimulus induces a vulnerable state by lowering sensory thresholds by the observation of a significant response to a second subthreshold stimulus delivered following resolution of pain from the initial injury27. The occurrence of generalized allodynia elicited by a subthreshold event may therefore represent PPTH-like behaviors.
Our studies using this model of PTH have shown that early peripheral CGRP signaling mediates APTH-like behaviors, the loss of DNIC in mice exposed to mTBI. Additionally, blockade of CGRP signaling early after the mTBI prevented the development of persistent PTH, with a more prominent role during the acute phase of PTH13,14. This model, therefore, may have significant translational relevance and may be valuable for future preclinical studies of pathophysiological mechanisms associated with APTH and PPTH. Additionally, this model may allow the exploration of novel mechanism-based therapeutic approaches. Thus, this protocol aims to describe in detail this unrestrained weight drop model of mTBI.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Clear PMMA chambers (10 x 10 x 20 cm)</td>
			<td>NA</td>
			<td>NA</td>
			<td>in house</td>
		</tr>
		<tr>
			<td>Disposable underpad</td>
			<td>VWR</td>
			<td>56617-014</td>
			<td>Can be replaced by any brand of laboratory disposable underpad</td>
		</tr>
		<tr>
			<td>Fluriso Isoflurane</td>
			<td>Vet One</td>
			<td>502017</td>
			<td>Can be replaced by any animal laboratory isuflurane inhalational anesthesia</td>
		</tr>
		<tr>
			<td>Inhalational anesthesia station</td>
			<td>Parkland Scientific</td>
			<td>PM1002</td>
			<td>Can be replaced by any animal laboratory inhalational anesthesia station</td>
		</tr>
		<tr>
			<td>LED lights for BLS exposure</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mesh stand (0.635 cm&#178; grid) for behavioral testing</td>
			<td>NA</td>
			<td>NA</td>
			<td>in house</td>
		</tr>
		<tr>
			<td>Mono nylon filament for hindpaw region (0.6 g/3.84)</td>
			<td>North Coast medical</td>
			<td>NC12775-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Mono nylon filament for periorbital region (0.4 g/3.61)</td>
			<td>North Coast medical</td>
			<td>NC12775-06</td>
			<td></td>
		</tr>
		<tr>
			<td>mTBI/Sham apparatus</td>
			<td>NA</td>
			<td>NA</td>
			<td>in house</td>
		</tr>
		<tr>
			<td>mTBI/sham induction-recording sheet of paper</td>
			<td>NA</td>
			<td>NA</td>
			<td>Prepare the sheet according to the Supplementary Material</td>
		</tr>
		<tr>
			<td>Pen</td>
			<td>Fisher Scientific</td>
			<td>NC1836221</td>
			<td>Any brand of pen</td>
		</tr>
		<tr>
			<td>Scotch Magic Clear tape 3M</td>
			<td>Fisher Scientific</td>
			<td>50-205-2453</td>
			<td>Can be replaced by any brand of clear tape</td>
		</tr>
		<tr>
			<td>Stopwatch</td>
			<td>Fisher Scientific</td>
			<td>06-662-56</td>
			<td>Can be replaced by any brand of stopwatch</td>
		</tr>
		<tr>
			<td>Tissue paper</td>
			<td>Kleenex/Fisher Scientific</td>
			<td>15-235-130</td>
			<td>Can be replaced by any brand tissue paper (4.4&#8221; x 8.4&#8221;)</td>
		</tr>
	</tbody>
</table>

unfixed head and closed skull weight drop method as a rodent model of mild traumatic brain injury (mtbi) for studies of post-traumatic headache (pth)

Mild traumatic brain injury (mTBI) represents one of the major contributors to the overall global disability. Post-traumatic headache (PTH) is a frequent and often the most debilitating associated symptom. For some patients, headache resolves in the first weeks to 3 months following a mTBI, a condition defined as acute PTH (APTH). However, several individuals continue to experience ongoing or intermittent headaches that persist beyond 3 months, a condition defined as persistent PTH (PPTH). Many animal models of TBI have been developed. Often, these are performed by impacting the head of rodents whose motion is mechanically fixed and/or involves an open skull. The following protocol describes a model where the head is unfixed, allowing free linear and rotational forces induced by a standardized weight drop that has similarities to concussions that occur in many situations in humans. The headache in this model is characterized by initial transient tactile allodynia in the cephalic region that lasts for several days and reflects the pain-like feature of APTH. Tactile allodynia is also observed in the extra-cephalic body areas suggestive of central sensitization and PTH. Following resolution of the transient APTH, animals display long-lasting latent sensitization, where baseline pain responses are normal, but otherwise innocuous provocative stimuli provoke cephalic and hind paw tactile allodynia, suggesting the development of persistent sensitization that may underlie PPTH. The key advantages of this model include concussion-like injury dynamics (closed head injury, reproduction of both linear and rotational forces) and induction of transient pain-like features followed by a long-lasting (persistent) period of increased vulnerability to normally innocuous stimuli that may reinstate a headache-like response. This method may be used to investigate the underlying pathological mechanisms involved in the transition of APTH to PPTH and the evaluation of novel therapeutic strategies for the treatment or prevention of APTH and PPTH.

Female (Figure 4) and male (Figure 5) mice were submitted to mTBI or sham injury. Development of immediate and transient periorbital (cephalic) (Figure 4A and Figure 5A) and hind paw (extra-cephalic) (Figure 4C and Figure 5C) CA was observed only in mice submitted to mTBI, demonstrated by increased frequency of response after tactile stimulatio...

The protocol describes a rodent model of unfixed head mild traumatic brain injury induced by a weight drop allowing for free linear and rotational impact-related forces. The dynamics of this injury are similar to concussions that occur in humans and are useful to investigate underlying mechanisms involved in post-traumatic headache.

Unfixed Head and Closed Skull Weight Drop Method as a Rodent Model of Mild Traumatic Brain Injury (Mtbi) for Studies of Post-Traumatic Headache (PTH)

Mild traumatic brain injury (mTBI) represents one of the major contributors to the overall global disability. Post-traumatic headache (PTH) is a frequent ...

Unfixed Head and Closed Skull Weight Drop Method as a Rodent Model of Mild Traumatic Brain Injury (Mtbi) for Studies of Post-Traumatic Headache (PTH)

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