A Cortical Injury Model in a Non-Human Primate to Assess Execution of Reach and Grasp Actions: Implications for Recovery after Traumatic Brain Injury

Abstract

Background:

Technological advances in developing experimentally controlled models of traumatic brain injury (TBI) are prevalent in rodent models and these models have proven invaluable in characterizing temporal changes in brain and behavior after trauma. To date no long-term studies in non-human primates (NHPs) have been published using an experimentally controlled impact device to follow behavioral performance over time.

New Method:

We have employed a controlled cortical impact (CCI) device to create a focal contusion to the hand area in primary motor cortex (M1) of three New World monkeys to characterize changes in reach and grasp function assessed for 3 months after the injury.

Results:

The CCI destroyed most of M1 hand representation reducing grey matter by 9.6 mm³, 12.9 mm³, and 15.5 mm³ and underlying corona radiata by 7.4mm³, 6.9mm³, and 5.6mm³ respectively. Impaired motor function was confined to the hand contralateral to the injury. Gross hand-use was only mildly affected during the first few days of observation after injury while activity requiring skilled use of the hand was impaired over three months.

Comparison with Existing Method(s):

This study is unique in establishing a CCI model of TBI in an NHP resulting in persistent impairments in motor function evident in volitional use of the hand.

Conclusions:

Establishing an NHP model of TBI is essential to extend current rodent models to the complex neural architecture of the primate brain. Moving forward this model can be used to investigate novel therapeutic interventions to improve or restore impaired motor function after trauma.

1.0. Introduction

Traumatic brain injury (TBI) is a major cause of death and disability. In 2014 the Centers for Disease Control and Prevention reported 2.53 million TBI-related injuries in the United States requiring 288,000 hospitalizations.¹ Because the number of TBI related deaths has decreased since 2001 due to advances made in acute management of TBI^2,3 many more TBI patients are surviving but with life-long disabilities. Efforts to improve daily lives of these patients are progressing by developing new translational models of TBI that focus on a variety of injury-related comorbidities. New studies are examining various aspects of TBI regarding initial severity and secondary outcomes such as neurodegeneration and delayed pathology related to cognitive and emotional aspects of TBI injury including an increase in studies assessing subsequent motor impairment. Variation in models range from global injuries to detect molecular disruption throughout the brain to more focal injury to assess memory or movement impairments as they apply to restorative therapies. Many investigators use focal injuries to reduce the range of complications of TBI with the intention of eventually addressing more generalized impairment with diffuse injury models.^4,5 One of the most common approaches for inducing focal impact injuries is the controlled cortical impact (CCI) model which allows reasonable consistency in the size and severity of injury.⁶ However, to date, most CCI studies utilize rodent species, primarily rats and mice. As CCI injuries have rarely been applied to larger animal models, it was our goal to extend this common focal injury approach to a non-human primate (NHP) species.

Most of the technical aspects for establishing parameters for reliable and valid implementation of experimental brain injury have been developed in rat models. Specifically, the Long Evans rat has been a reasonable model for these studies, and we have developed reliable methods for creating repeatable CCI injuries in motor cortex in this strain and have tested novel therapeutic approaches.^7–9 However, rodents have limitations with regard to translation to clinical studies. As novel restorative therapies are emerging from the rodent literature,¹⁰ development of additional translational models is needed to establish feasibility and efficacy in more complex mammalian nervous systems such as those of NHPs.^11–14 To date, CCI injury models in NHPs have only been established for short-term (< 2 days) survival studies, and thus, behavioral recovery data in NHPs after CCI injuries are non-existant.^15,16

NHP models of brain injury are well suited to address multiple neural mechanisms capable of supporting recovery, providing a potential model for rehabilitative or restorative therapies.^17–19 As we have had several decades of experience with focal ischemic lesions in motor cortex of squirrel monkeys (Saimiri spp.), ^20–30 the present study was designed to test the feasibility and reproducibility of CCI injuries in squirrel monkeys. An advantage of this species for initial characterization of this model is that the somatosensory and motor areas are easily accessible on a flat, relatively unfissured sector of frontal cortex. Also, squirrel monkeys have well developed parietal-frontal networks related to visuomotor control of the forelimb and hand mediated through several premotor cortical areas unique to primates.³¹ The complex neurophysiological interaction within the visuomotor system of primates, offers a unique flexibility of muscle recruitment to achieve behavioral goals³² guiding the development of novel therapies that can later be tested in human cohorts. The present study is unique with regard to the long-term (3 months) assessment of motor recovery after a CCI injury in an NHP.

2.0. Methods

Three male squirrel monkeys (Saimiri spp., weight range 828 – 1102 g) were used to model a focal concussive injury restricted to the distal forelimb area (DFL) of primary motor cortex (M1). All procedures were conducted in accordance with institutional and federal guidelines for care and use of experimental animals and with approval from the Institutional Animal Care and Use Committee. Monkeys were housed in a climate-controlled vivarium. Food (Purina Lab Diet #5088) was provided twice daily (supplemented with fruit and vegetables) and water was provided ad libitum through an automatic watering system. On days when behavior was assessed, feeding was scheduled after assessment. Routine health checks were made daily by Laboratory Animal Resource staff under supervision of the institutional veterinary staff.

2.1.0. General Procedures.

Each monkey received a focal concussive injury after craniectomy over the M1 DFL using the CCI procedure in either the left or right hemisphere (contralateral to their preferred hand used on a dexterity task). The DFL area was delineated using standard intracortical microstimulation (ICMS) procedures to delineate proximal and distal forearm movements.^20–30 Behavioral assessments of dexterity and power grip were obtained over five sessions distributed over two weeks prior to the CCI, and once per week over the following three months after injury. Neural recordings were acquired within and adjacent to the cortical impact area in two monkeys at the end of behavioral assessment in a final procedure to verify the effectiveness of the injury (3 months post-CCI). All monkeys were humanely euthanized at the end of the experiment followed by transcardial perfusion and fixation with 10% Formal saline.

2.1.1. Behavioral Procedures.

General methodology.

Two different behavioral tasks were used to assess dexterity and hand grip performance, respectively (Figure 1). In both behavioral tasks, monkeys were reinforced with a food reward for successful performance. First, a modified Klüver board was used to determine hand preference, and to assess dexterity by presenting food-pellets to the monkeys on a flat manually rotated disk with wells of different size for placement of banana flavored food pellets, as described in more detail below. Second, a hand grip device was used to assess complex and simple use of the power grip. The hand grip device was programmed to two different sets of criteria, providing the means to assess power grips on either a “Difficult Task” or an “Easy Task” as described below. To obtain a food reward, the monkey was required to squeeze a handle embedded with a calibrated force transducer, applying a target force range for a specified time. If the criteria were met for an individual grip, a food-pellet was delivered to a food bin placed within the monkey’s cage when the handle was released. Learning the association between gripping the handle and receiving a reward was more challenging for the monkeys than extracting food pellets from the food-wells, and thus required multiple sessions for consistent performance.

Figure 1 A - B.

Motor skill assessment apparatus. A. Klüver Disk Task. This task was used to train and assess skilled use of grasping during a reach and retrieval task. Monkeys were required to retrieve a single 45 mg food pellet (3.56mm diameter) delivered singularly to five food-wells. Each food well was 5 mm deep and ranged in diameter from 9.5 mm to 25 mm. B. Hand Grip Task. This task was used to assess ability of monkeys to apply and sustain a power grip within a range of either 100 – 300 g of force for 3 seconds (“Difficult” task) or 20 – 500 grams of force for 0.5 seconds (“Easy” task).

Subsequent to learning each of the behavioral protocols, the behavioral tasks were presented to each monkey five days per week during single sessions in the order of most difficult to easiest task: “Difficult” hand grip task followed by the Klüver disk task and ending with the “Easy” hand grip task. This order was used to maintain motivation to work for food-pellets on each task. Pre-injury baseline performance was collected over five sessions. Natural recovery from injury was assessed beginning one week after the CCI injury and continuing once per week for 13 weeks. Behavioral assessments after injury were conducted weekly to minimize potential training effects. Behavioral sessions were videotaped for later analysis.

Klüver disk task.

This task was slightly modified from one used in several of our previous publications^20–23 to accommodate the food wells on a circular disk (Figure 1A). A circular Plexiglas disk attached to a frame and mounted onto the front of the monkey’s home cage, with five food wells drilled into its surface. The frame incorporated a sliding door that restricted retrievals to the preferred hand both before and after injury. The disk could be rotated to present each food well to the same position in front of the monkey. Each well was 5 mm in depth and ranged in diameter from 25 mm to 9.5 mm (designated wells 1–5 respectively): Well-1 = 25 mm dia.; Well-2 = 19.5 mm dia.; Well-3 = 13 mm dia.; Well-4 = 11.5 mm dia.; Well-5 = 9.5 mm dia.. The Klüver disk task consisted of reaching through the cage bars, inserting one or more fingers into a food well, and retrieving a single, small banana flavored food pellet (45 mg; BioServe, Flemington NJ). Food pellets were placed one at a time into one of the five food wells in a randomized block design (i.e., equal number of presentations for each well). Dexterity was measured as the number of digit flexions necessary to remove food pellets presented per well on each session. There were three steps to establishing baseline performance on the Klüver disk task: (1) acclimation/hand preference assessment; (2) skill training on all food-wells; and (3) random probe trials on all food-wells. Prior to training, each animal was acclimated to the Klüver disk and assessed for hand preference over 50 trials a day on two consecutive days while reaching and grasping food pellets with either hand without restriction. Hand preference was determined as the hand used for over 50% of the trials. A sliding Plexiglas door was installed on the frame to constrain use of either the right or left forelimb. Training was conducted with each animal’s preferred hand over ten consecutive days for 30 min per day beginning with the largest well (Well-1) and ending with the smallest well (Well-5) on the 10^th day. Wells 4 and 5 had the smallest diameter and were the most difficult for the monkeys to extract the food-pellets. To encourage motivation, a progressive training schedule was instituted. The timeline for training on the Klüver disk was as follows: Day 1: Well-1; Day 2: Well-2; Day 3: Well-3; Day 4: 50% Well-3/50% Well-4; Day 5: Well-4; Day-6: 50% Well-4/50% Well-5; Day-7 through Day-10: Well-5. Probe trials were used to maintain performance while training began with the grip-tasks (see methods below). The probe trial session consisted of randomly presenting 10 food-pellets per well to each animal’s preferred hand. Baseline performance was based on the last five sessions prior to CCI.

Hand-grip task.

The grip device consisted of an aluminum cylinder connected to a universal joint allowing the cylinder to rotate in multiple directions, as described in an earlier publication from our laboratory (Figure 1B).³³ The cylinder and u-joint (grip handle) were attached to an aluminum frame mounted to the monkey’s home cage, suspending the grip handle in front of the monkey. The grip handle was attached to a rail running along the length of the frame and could be adjusted to each monkey’s hand preference. A sliding Plexiglas door was used to limit the monkey to use of either the left or right hand based on hand preference on the Klüver task. The combination of the u-joint and the sliding grip handle helped to minimize the use of proximal musculature during the power grip³³. Grip force was measured by a Honeywell Sensotec Compression Load Cell with a 1,000 g limit and 0.01 g resolution (Honeywell Sensotech, Columbus Ohio). The load sensor was checked for calibration prior to each session. The grip task required each monkey to exert and hold a grip force between a low and high cutoff point over a specified period of time to receive a food pellet reward. A LabView program was used to acquire grip information and set parameters used to assign successful grip instructions necessary to achieve a food reward. Training began with an initial reward criterion of 25 to 500 g force for 0.5 sec. Training proceeded by increasing the lower limits of grip force while lowering the upper limits in increments of 25 g while increasing hold time by 0.5 sec. When 60 pellets were obtained, the reward thresholds were changed until the monkey could attain 100 – 300 g force for 3 sec. To obtain a food reward, the monkey was required to squeeze the handle activating a force transducer, applying a specified range of force and time defined for the “Difficult” and “Easy” task. Food-pellets were delivered to a food bin placed within the monkey’s cage once the handle was released. The monkey could make as many grips as possible during a 30-min session on the “Difficult” task and during a 5-min session on the “Easy” task.

2.1.2. Behavioral data analysis.

Hand dexterity was assessed as the number of digit flexions necessary to retrieve food pellets from the Klüver disk. Grip performance was assessed as the number of successful grips made during a 30-min session on the “Difficult” task and during a 5-min session on the “Easy” task. Each monkey’s individual performance score on a given day was compared to that monkey’s distribution of pre-injury baseline performance over five sessions. Each monkey’s average baseline success and standard deviation was used to establish a 95% confidence interval (CI; Z = 1.95) used to determine a significant deviation from pre-injury baseline performance.

2.1.3. Surgical Procedure.

Sterile surgical procedures were used to expose the M1 DFL and the surrounding cortical territory to identify movement representations as defined by ICMS techniques. Monkeys were sedated with an initial dose of ketamine (20 – 30 mg/kg, i.m.) followed by atropine (0.07mg/kg i.m.). Supplemental doses of ketamine (20 – 30 mg/kg i.m.) were administered as needed as the monkeys were weighed and examined in preparation for the surgical procedure. The scalp, forelimb (opposite the cerebral hemisphere to be mapped) and hindlimb (for catheterization) were shaved and cleaned. The trachea was treated with a 20% benzocaine spray followed by 2% lidocaine jelly while intubated (2.5–3.0 mm i.d. tracheal tube). Following surgical preparation, isoflurane (0.5 to 3%) was introduced and the saphenous vein catheterized with a 24-gauge angiocath for delivery of lactated Ringers with 3% dextrose (10ml/kg/hr) and other required fluids. Ophthalmic ointment was liberally applied to the corneas and the monkey was placed into a stereotaxic frame. The incision area was infiltrated with 0.5% bupivacaine (1 ml s.c.) and scrubbed with alcohol and iodine. Penicillin-G benzathine + procaine (45K IU, s.c.), dexamethasone (0.50mg i.v.) and mannitol (2g/kg i.v.) were administered.

Under aseptic conditions and 1.5% – 3% isoflurane- 75% nitrous oxide anesthesia, a craniectomy (~1.5 cm²) was performed over M1 and extended to include S1 and premotor cortex contralateral to the monkey’s preferred hand. A sterile plastic chamber was temporarily secured over the opening and filled with sterile silicone oil to protect the cortex during the ICMS procedure and removed prior to the CCI procedure. Inhalation anesthesia was withdrawn, and ketamine (5mg dose), supplemented by diazepam (0.01mg per dose), was administered intravenously as needed. Vital signs were monitored and maintained within normal limits throughout the procedure. Heart rate, saturated oxygen, respiration rate and expired CO₂ were monitored with a gas monitoring system and core body temperature was monitored and maintained with a homothermic blanket system. Following an ICMS mapping procedure identifying DFL (see below), inhalation anesthesia was reinstated, and the dura replaced over the cortex. A CCI was then delivered (see below). The dura was then removed and replaced with a thin silicone sheet. Dental acrylic was used to replace the craniectomized bone over M1, and the incision was sutured and treated with 0.5% bupivacaine (1ml s.c.).

Post-operative support included penicillin-G benzathine + procaine (45K IU, s.c.), topical triple antibiotic and analgesics acetaminophen (10–20 mg/kg) /codeine (1 – 2 mg/kg) solution (oral) and tramadol (3 mg/kg, i.m.). Supplemental support was given as needed determined by veterinary consultation. Monkeys were monitored in a temperature-controlled incubator until recovery from anesthesia was complete and the animal was alert and stable (indicated by mobility, eating/drinking, waste elimination), and then returned to their home cages. Prolonged care procedures included wound care as needed, suture removal at ~2 weeks post-surgery and daily monitoring of general health.²⁹

A final surgical procedure was performed as described above at the experimental end point (3 months post-CCI) to assess damage to cortex within and around the impact. A digital photomicrograph was taken of the cortical surface and used to assess extent of the cortical damage. Neural recordings were acquired within the M1 infarct area and in the adjacent premotor cortex. Monkeys were humanely euthanized at the end of the final procedure.

2.1.4. ICMS Procedure.

A cortical mapping procedure was used to identify the distal forelimb representation (digits, wrist and forearm) and its borders with the proximal forelimb (shoulder, elbow) and face representations. Movements were evoked by delivering electrical current through a stimulating microelectrode (~15 μm tip o.d.) placed perpendicularly to the cortical surface, targeting layer V at a depth of ~ 1750 μm. The microelectrode consisted of a platinum wire insulated by a glass micropipette filled with a 3.5 M NaCl solution. Current was delivered from a BAK stimulus isolator (Model BSI-2, BAK Electronics Inc.) as a 45 msec pulse train consisting of 13 cathodal monophasic pulses (0.2 ms duration; 3.3 ms between pulses; capacitively coupled), with pulse trains delivered once per sec. The stimulus pulse was programed via a Master-8 stimulus pulse generator (A.M.P.I., Isreal), and the current waveform was monitored on an oscilloscope. Maximum current did not exceed 30 μA.

2.1.5. CCI Procedure.

A commercial electromagnetic impact device was used to deliver a traumatic brain injury to the M1 DFL (Impact One, Leica Biosystems, Buffalo Grove, IL). Parameters for the CCI were entered into a control unit attached to a linear actuator mounted on a micropositioner. The arm of the micropositioner was modified with a universal joint to align the impact tip perpendicular to the cortical surface. Conductance through the impact tip allowed calculation for the depth of impact from the surface of the cortex. The impact parameters were based on previous studies using CCI procedures.^8,15,34 The impact tip was a stainless-steel rod with a 5 mm diameter tip (slightly beveled to remove sharp edges around the perimeter). The impact tip was aligned over the center of the M1 DFL as delineated with ICMS and recorded onto a photomicrograph of the cortical surface. The boundary of the DFL was aligned relative to the surface blood vessels. The impact was delivered with a depth of 3 mm from the cortical surface, at 4.5 m/sec with a dwell time of 200 msec.

2.1.6. Neurophysiological Procedure.

Three months after a CCI injury in M1, neural firing rates were acquired for two of the three monkeys (A385 and A401) under anesthesia (70% NO₂ and 1.5 % isoflurane). A 16-channel, single-shank acute planar probe (NeuroNexus 1×16–713-200) was connected to a unity gain headstage and signal passed to a digitizing amplifier (Tucker-Davis Technologies, Alachua, FL) sampled at 24,414 Hz. An online, adaptive threshold crossing was set at 5.5 SD of root mean square (RMS) of the recorded signal per channel in 5 sec moving windows. The timestamps of all threshold crossings were recorded and used to calculate a per-channel mean firing rate (MFR) for multiunit activity over the entire duration of the recording trial (typically 5 min per site). Samples were acquired from i) the impacted M1 lesion area, ii) areas along the anterior and posterior border of the M1 lesion area and iii) more anteriorly in the premotor area (PM). A natural log transformation was used to normalize spiking rates (spikes per second) for parametric analysis. A one-way ANOVA was used to compare the three sampling areas followed by protected t-test to compare neural activity within PM to activity within lesion core and lesion borders.

We also mapped somatosensory cortex (S1) to delineate the cutaneous representations of digits 1 and 2 for reference. A Michigan-style electrode (impedance ~ 700 kΩ; NeuroNexus, Ann Arbor, MI) was lowered perpendicular to the cortical surface to span cortical layers II-V. Signals were filtered and amplified using a commercial neurophysiological recording system (Tucker Davis Technologies, Alchula, FL) and played over a loudspeaker for monitoring. Receptive fields were determined by neural responses in S1 to light touch with a Semmes-Weinstein monofilament 3.61 (~400 mg force). Deep receptive fields were defined by higher threshold stimulation when cutaneous responses were not detected (>400 mg force) and manipulation of joints.²⁶ Receptive fields through the four subregions (Areas 3a, 3b, and 1 / 2) were identified and plotted on a digital photograph of the cortical surface.

2.1.7. Histological Procedure.

Monkeys were humanely euthanized with a combination of dexdormitor 100 mcg/kg i.m. followed by Beuthanasia-D 1.5 cc i.p. The animals were then perfused through the left ventricle of the heart with heparinized saline and lidocaine followed by 10% Formal saline.

Analysis of injury extent on cortical surface.

Pre- and post-CCI digital photographs of the cerebral cortex were compared to identify the lesion area. Pre-CCI cortical maps showing location of the M1 DFL area targeted for impact were superimposed onto the digital photograph of the cerebral cortex. This composite photograph with functional boundaries was compared to a digital photograph of the post-CCI cortical surface, and a graphics program (Canvas v12, ACD Systems of America) was used to measure the cortical extent of the lesion in mm². Changes in cortical surface area were identified relative to changes in blood vessel patterns and necrotic tissue.²⁹ Whole brains were fixed in 10% formalin and stored in 0.1 M sodium phosphate buffered saline (PBS) and then shipped to Neuroscience Associates (NSA, Knoxville Tenn.) for histological processing. Coronal sections were cut at 40 μm thickness. In each animal, we used 42 Weil stained sections (80 μm separation between sections) through the impact area (5 mm) and adjacent tissue to evaluate the change of volume limited to the corona radiata. The contour of gray matter, white matter and the injured boundaries in each section were visualized with the Weil stain and traced under 2.5X magnification using a quantitative microscopy system (Neurolucida; MBF-Bioscience version 11). The volumes of the corona radiata under the impact area of cortex in injured and intact hemispheres were estimated according to the Cavalieri principle using the section cut thickness (40 μm), a slice interval of 3, and a 400 μm grid spacing (StereoInvestigator, MBF-Bioscience, Version 11).

3.0. Results

3.1. Lesion Extent.

In each of the three monkeys, the cortical territory at the site of impact (Figure 2A, B) appeared shrunken three months after the CCI (Figure 2B) that was clearly visible as a necrotic area in the perfused brain (Figure 2A). Comparison of pre-CCI and three-month post-CCI photographic images (Figure 3A) revealed a reduction in impacted M1 DFL cortex for each monkey exceeding 70% of the impacted area: 73.6%, 76.7% and 85.6% in A401, A385 and A413, respectively (Figure 3B). Myelin-stained sections show that in each of the three monkeys, the lesion was almost exclusively confined to the cortical grey matter, with minimal to no intrusion into the underlying white matter (Figure 3C). Estimated volume of white matter within the corona radiata beneath M1 DFL indicated a small loss of fibers in each monkey. The volume of corona radiata underlying the injured cortex was reduced by 5.29%, 4.10%, and 11.66% in A401, A385 and A413, respectively.

Figure 2 A - C.

Verification of lesion 3 mos post-CCI. A. Post-mortem photograph of the perfused brain of monkey A401 three months after a CCI injury. The CCI impact is seen within the red circle on the photograph. The blue circle indicates the position of the craniectomy made during the initial procedure to access cerebral cortex for identifying M1 and adjacent motor and somatosensory areas. Dark red areas in the photograph along the blue outline resulted from residual blood around perimeter of craniectomy that did not wash out during postmortem perfusion. B. Interaoperative photograph of the cerebral cortex within the cranial opening taken three months after CCI injury showing sites where neural recordings were acquired. Large red outline shows distortion of impacted cortex three months after CCI. Neural recordings were acquired from intact cortex adjacent to injury (large blue filled circles) and from infarct area (large black filled circles). Cutaneous responses were also obtained in primary somatosensory cortex. Topographical organization of the hand responsive to cutaneous stimulation was sampled in somatosensory cortex. The resulting cutaneous hand-map appeared normal throughout areas 3a, 3b, and area 1 / 2 outlined on the photograph. Sites where neural responses were evoked from cutaneous stimulation of the digits are shown as small red filled circles; glabrous cutaneous responses along the hand are shown as small white filled circles outlined in black. C. Neural spike recordings from inside impact area labeled “1” and outside impact area labeled “2”. Neural activity during a 1 second sample is shown for 16 channels acquired from depths of 200 to 1,700 μm (left side of each figure). Very little spontaneous spiking activity was observed within the impact area (Site 1) compared to an adjacent non-impacted site (Site 2), indicating that the lesion was effective throughout the depths of the grey matter. Mean firing rate (MFR) ± SE = 0.16 ± 0.0023 spikes/sec across 16 channels in Site 1 and 9.5 ± 1.15 spikes/sec in site 2.

Figure 3.

A. Intraoperative photographs of exposed cortex before and after CCI. Solid red outline identifies target for CCI before impact in Pre-CCI column whereas a dashed red circle identifies the corresponding cortical surface (based on vascular patterns) three months after impact in Post-CCI column. B. Cortical area under impact was reduced by 85.6% (16.8 mm²), 76.7% (15.1 mm²) and 73. 57% (14.5 mm²) in A413, A385 and A401, respectively. C. Coronal Sections (Weil stain). First column shows photos (2.5X) of coronal sections through the impacted area in each of the three monkeys. Second column is a magnified view of the location of impact. The Weil-stained sections were used to estimate area of the corona radiata underlying the area of cortical infarct.

Three months after the CCI injury, neural recordings were collected within, and adjacent to, the impact area to compare spiking activity in the targeted M1 DFL and in adjacent perilesion areas anterior and posterior to the impact (Figure 2B, C). A total number of 96 sites were recorded for A385: 32 within M1, 16 on rostral and 16 on caudal border of M1 and 32 in PM. A total number of 80 sites were recorded for A401: 16 within center of M1 lesion area, 16 on rostral and 16 on caudal borders and 32 within PM. Both A385 (MFR ± SE: 0.39 ± 0.38 Hz) and A401 (0.16 ± 0.0023 Hz) showed little neural activity within the lesion core. A385 had increased MFR along the border of the lesion core (1.53 ± 0.38 Hz); A401 showed a smaller MFR along the border (0.077 ± 0.0018 Hz). MFR were also acquired from PM for A385 (0.75 ± 0.16 Hz) and A401 (6.6 ± 0.84 Hz). A one-way ANOVA found significant differences among the 3 areas for A385 (F_(2,93) = 15.65; p<0.0001) and A401 (F(2,77)= 377.9; p<0.0001). A protected t-test comparing PM neural activity for A385 and A401 found significant differences between PM and CCI core activity for both monkeys: A385 (t(1,93)= 2.36; p=0.02) and A401 (t(1,77)= 17.66; p<0.0001). Figure 2 shows neural activity from one sampling area within the impact zone demonstrating less spontaneous spiking activity than a sampling area outside of the impact zone.

3.2. Behavioral Assessment on Klüver Disk Task.

In the initial days following the lesion, each monkey exhibited disuse of the hand contralateral to the CCI injury that resolved prior to behavioral assessments. Within the first week after CCI, each monkey regained use of both hands for grooming and feeding while moving around their home cage, grasping cage bars, climbing and balancing on their perch.

The ability to successfully reach and retrieve food pellets from each of the five food-wells varied among the three monkeys after the CCI injury (Figure 4). Two of the three monkeys (A413 and A401) could not successfully retrieve a food pellet from any of the wells initially after the CCI. A413 was able to retrieve food pellets from well 4 by the second week after the injury but could not retrieve from well 5 (smallest well) until 7 weeks after the injury. A401 was able to perform the task on wells 4 and 5 by the third week after the injury. Difficulty in grasping and retrieving food pellets from the food-wells persisted as indicated by the number of finger flexions required to extract and retrieve a food pellet. Impairment in extracting food pellets lasted through the entire three months of post-CCI assessment. Even though extracting the food pellets from the larger food wells (wells 1 to 3) was affected by the impact, all monkeys could extract food pellets with one or two flexions by the third week (well 1–3 data not shown). Impaired motor skills were more pronounced for extracting food pellets from the smallest wells (well 4 and well 5).

Figure 4.

Hand dexterity as assessed by the Klüver Disk Task. Motor performance (digit flexions) was determined relative to each monkey’s average pre-injury baseline distribution of scores over five sessions collected over a two-week period prior to CCI injury. Following CCI injury, flexions per retrieval scores on a given weekly session (1 to 13) were converted to z-scores standardized to the mean and standard deviation of the individual monkey’s baseline distribution. Scores falling outside of a 95% CI (z=2.0) were considered significantly different from baseline. The 95% CI is designated by the grey shaded area. Scores outside of the shaded area are outside of the 95%. Weeks prior to CCI injury are indicated as negative numbers.

3.3. Behavioral Assessment on Hand Grip Task (Figure 5).

Figure 5.

Hand grip performance. Number of successful grips on a given weekly session (1 to 13) was converted to z-scores standardized to the mean and standard deviation of the individual monkey’s baseline distribution. The 95% CI for each monkey is designated by the grey shaded area. Scores falling outside of the shaded area (z=2.0) were considered significantly different from baseline. All monkeys were impaired to varying degrees on the “Difficult” task but only a mild transient deficit was observed on the “Easy” task.

Easy task (25 – 500 g for 0.5 sec).

Prior to the CCI injury, each of the three monkeys was able to perform the Easy task. However, there was substantial session-to-session variability in the number of successful grips, especially in A413. After the injury, each of the three monkeys was able to perform the Easy grip task throughout the post-injury period. However, the number of successful grips per session occasionally fell below the pre-lesion baseline range (> 2 std dev). Significantly fewer successful grips were observed for A413 on post-injury week 2, A385 on week 1, and A401 on week 7.

Difficult task (100 – 300 g for 3 sec).

Prior to the CCI injury, each of the three monkeys was able to perform the Difficult grip task, which required a more restricted force range for an extended time period compared with the Easy task. Prior to CCI injury, task acquisition varied among the three monkeys learning to hold a grip between 100 – 300 g for 3 sec: A401 required 50 sessions, A413 required 46 sessions and A385 required 35 sessions. Performance on the Difficult grip task also varied among the three monkeys after CCI injury. During the first week after CCI each of the monkeys had difficulty reaching the 100 g threshold and maintaining a grip between 100 and 300 g for 3 sec. Each of the monkeys was able to apply grip force within the baseline range (95% CI of their respective baseline performance) within 5 weeks. However, performance of A385 was highly variable and declined by week 6 relative to baseline. Grip performance in this animal generally remained below baseline levels through the remainder of the 13-week study. The other two monkeys (A401 and A413) remained within their 95% CI from week-6 to week-13 and performing consistently within the baseline range by the end of the study.

4.0. Discussion

Over the last few decades, the intrinsic capacity of the mammalian brain to reorganize after injury and restore motor function has been demonstrated with various animal models.^35–37 Translational research identifying adaptive molecular mechanisms initiated by injury and subsequent anatomical reorganization are leading to new innovative therapies to restore neurological function.³⁶ A major benefit of using animal models is the control of variability in size and location of injury to address specific mechanisms underlying consequential neuropathology and recovery.³⁷ This depends upon controlling the specificity of a brain injury to isolate the contribution of distinct neural systems in the development of subsequent anatomical and physiological disruption and potential resolution of function (e.g., cognitive, motivational and motor). Improvements in cortical impact devices such as the programmable CCI impactors currently in extensive use have allowed for necessary experimental control to examine various aspects of traumatic brain injury.³⁴ Choosing the most appropriate species is also necessary when assessing reorganization of complex neural systems and behavioral outcomes. In this study we present an NHP model of TBI to assess deficits in motor performance and ultimately, subsequent changes in functional neural connectivity responsible for the recovery. The evolution of cortical specializations in primates results in unique sensorimotor integration patterns between anatomically separated cortical areas necessary for the complex behavioral demands of their environments.^{38, 39}

This initial study to introduce an NHP model of TBI focuses on motor recovery in the use of the forelimb, characterized disruption of grasping performance during natural recovery from a CCI injury to the DFL (including the hand) representation in M1. We chose a focal M1 injury for our initial study because of the multiple corticocortical loops with premotor, somatosensory and parietal cortices, contributing to the complexities of motor control of skilled use of the hand, and because of the wealth of data from our laboratory on the effects of ischemic infarcts in M1. In primates, multiple hand areas exist in remote locations within motor and sensory cortex. Each hand area in premotor and somatosensory cortex communicates directly with M1.^{39 – 41} Injury to one or more specialized hand areas such as M1, ventral premotor cortex, dorsal premotor cortex or the supplementary motor area, leads to reorganization of activity within areas not directly injured and this reorganization is associated with behavioral recovery. ^{26, 27, 42}

In the present study, an impact confined to the DFL representation of M1 in squirrel monkeys impaired volitional, goal directed movements requiring skillful manipulation of digits and sustained grip. Typical behaviors such as feeding, grooming, and arboreal locomotion were only transiently affected contralateral to the CCI, recovering within the first week after injury. This is similar to what we have observed after a focal ischemic infarct in the same cortical location in this species.²⁴ Throughout the following weeks of recovery after the CCI, moderate motor impairments were observed during performance on two tasks that required skilled use of the distal forelimb learned prior to injury. Thus, in general, the CCI model was quite successful in the squirrel monkey, using impact parameters similar to those used in rodents. Effective lesions of similar size were produced, and the injuries resulted in moderate motor impairments. Also, the damage was largely confined to the cortex with minimal damage to the underlying white matter.

The Klüver disk task required squirrel monkeys to retrieve food pellets from various sized food-wells, usually by bracing some of their digits or palm against a Plexiglas board while manipulating a small food pellet with their free digits. This is a learned motor skill for squirrel monkeys.²³ With practice, uninjured monkeys can learn to retrieve food pellets with a single flexion from the smallest food well. After ischemic injury to motor cortex, the number of digit flexions needed to remove a food pellet increases.²⁴ Some improvement in skilled use of digits improved naturally over time (i.e., spontaneously or without rehabilitative training) but in two of the three monkeys (A413 and A385), grasping performance was still well below pre-injury baseline levels after 13 weeks of post-CCI assessment. Monkey A401 performed the Klüver disk task better than monkeys A413 and A385 prior to injury and showed better recovery over time. It is worth noting that this monkey also had the smallest lesion of the three monkeys.

The persistent impairment affecting coordinated use of digits during pellet retrieval is similar to those reported with patients diagnosed with pure motor hemiparesis as the monkeys were able to regain use of hand and digits but not fully recover function needed for skilled motor performance.⁴³ Typically, clinically identified pure motor hemiparesis is seen after lacunar stroke in the posterior limb of internal capsule, corona radiata, or corticospinal tract. Lang and Scheiber found that damage restricted to motor cortex and corticospinal tract would also result in a pure motor hemiparesis of the hand affecting finger movements but not cognitive aspects of movement.⁴³

Impaired use of the hand was also evident from the sustained grip task we implemented within a conditional association paradigm. The monkeys had to associate presentation of a grip handle with a food reward located at a distance from the grip handle under specific conditions defining the force and duration of the grip. The injury resulted in a less severe or transient impairment in gripping behavior in contrast to grasping behavior observed on the Klüver task. The ability to sustain a power grip between 100 to 300 g of force for 3 sec (“Difficult Task”) recovered to baseline for each monkey. Grip performance in two of the monkeys (A413 and A401) was indistinguishable from baseline performance by the 2nd month post-injury. The third monkey, A385, was much more variable, with sessions within the baseline range, interspersed with sessions with poorer than baseline performance. However, this monkey still successfully reached 46% to 86% of baseline performance on the “Difficult Task” over weeks 6 to 13. The association of grasping the handle to receive a reward was not substantially affected by CCI-injury as evidenced by the consistent performance on the “Easy Task”.

This study is unique in demonstrating the feasibility of the CCI approach to modeling long-term recovery after TBI in an NHP. However, the study has two notable limitations: First, while the relatively lissencephalic brain of the squirrel monkey aids greatly in the neurophysiological identification of somatosensory and motor areas for targeting the injury, the mechanical stress differs from more gyrencephalic brains. It has been suggested that mechanical injuries in gyrencephalic brains result in more brain deformation, especially at the base of the sulci.⁴⁴ In the more lissencephalic squirrel monkey brain, the mechanical stress, and hence the injury extent was largely limited to the cortical grey matter at the surface (Figure 3C). Second, the motor impairments in the current study were relatively mild and somewhat transient. However, an advantage of the CCI approach with programmable devices is that the impact parameters can be easily adjusted to suit the goals of the particular study. Alternatively, the injury location can be varied. In our particular model, it was important to select specific parameters for CCI to limit damage to the DFL area of M1, sparing premotor cortex and parietal-frontal connections related to behavioral goals and attentional control of movement. ^{45, 46}

These results emphasize the precision of the CCI model to produce a focal injury to target specific cortical areas such as M1 or premotor hand areas as needed. Moving forward, the NHP model will allow us to address complex interactions between specialized corticospinal connectivity related to corticospinal and corticobulbar projections from various premotor cortices that remain intact after the injury.^{47 – 51} Although there are many similarities between sensorimotor networks in rodent and primates, organization of primate motor systems reflect evolution of specialized visuomotor adaptations to arboreal environments.^{38, 52} Advances in clinical application and development of neuroprosthetics and brain-machine-brain interface devices depend on advancing models of primate cortical organization.

Highlights.

• First nonhuman primate model of controlled cortical impact assessing motor recovery

• A grip device was developed and programed in Lab View to assess sustained grip

• Motor impairment was more pronounced for skilled use of digits than sustained grip