Spinal cord injury transiently alters Meissner’s corpuscle density in the digit pads of macaque monkeys

Abstract

Meissner’s corpuscles (MCs) are cutaneous mechanoreceptors found in glabrous skin and are exquisitely sensitive to light touch. Along with other receptors, they provide continuous sensory feedback that informs the execution of fine manual behaviors. Following cervical spinal deafferentation injuries, hand use can be initially severely impaired, but substantial recovery occurs over many weeks, even when ~95% of the original input is permanently lost. While most SCI research focuses on central neural pathway responses, little is known about the role of peripheral receptors in facilitating recovery. We begin to address this by asking the following: (1) What is the normal pattern of MCs in the distal pads of all five digits in the macaque monkey (with hands similar to humans)? (2) What happens to these receptors 4–5 months following either a dorsal column lesion (DCL) or a combined dorsal root/dorsal column lesion (DRL/DCL), when functional recovery is largely complete? (3) What happens chronically, 12–14 months later?

Our findings show that in normal monkeys, MCs are densest in the distal pads of the opposing thumb and index finger, with the greatest concentration on the thumb. This reflects a close functional relationship between receptor density and precision grip. At 4–5 months post-injury, there was a (~30%) loss of MCs on the deafferented digits of the injured hand compared with the contralateral side. However, 12–14 months after a DRL/DCL, receptor densities had returned to normal levels. Our findings indicate a complex peripheral response and highlight the importance of the periphery in shaping central changes.

Graphical Abstract

Meissner’s corpuscle (MC) densities were compared across digits in normal and lesioned monkeys, to determine changes following cervical spinal deafferentation injuries. In normal animals (green), MCs were densest in opposing digits. Four months post-lesion (blue), densities in targeted digits had decreased significantly, but returned to normal 1 year later (purple).

Introduction

Spinal cord injury (SCI) is a devastating event that typically produces permanent sensory and motor loss below the site of injury. Although there has been extensive research into the mechanisms of recovery following SCI, this work has focused on changes in the central nervous system (Ballermann & Fouad, 2006; C. Darian-Smith, 2004; C. Darian-Smith, Lilak, & Alarcon, 2013; C. Darian-Smith, Lilak, Garner, & Irvine, 2014; Fisher, Lilak, Garner, & Darian-Smith, 2018; Jain, Florence, Qi, & Kaas, 2000; Rosenzweig et al., 2010; Weidner, Ner, Salimi, & Tuszynski, 2001; Zorner et al., 2014), and has largely ignored the peripheral response and its role in the recovery process.

In humans and nonhuman primates, fine motor skills are facilitated by cutaneous and proprioceptive sensory feedback (C. Darian-Smith & Ciferri, 2005; I. Darian-Smith, 1984; Glendinning, Cooper, Vierck, & Leonard, 1992; Gordon, Ghilardi, & Ghez, 1995; Nathan, Smith, & Cook, 1986; H.-X. Qi, Gharbawie, Wynne, & Kaas, 2013). Peripheral receptors in the hand receive and transmit sensory information to the somatosensory cortex, where it is used to direct coordinated motor output. Meissner’s corpuscles (MCs) are a type of cutaneous mechanoreceptor found in the glabrous skin of mammals, and are known to be particularly abundant in the fingertips. They are located just below the epidermis in the dermal papillae and are sensitive to light touch and vibration. Physiologically, they are rapidly adapting (RA) mechanoreceptors with small receptive fields (1–2 mm diameter) that encode transient changes in sensory input to the skin (Hoffmann, Montag, & Dominy, 2004; Zimmerman, Bai, & Ginty, 2014). Along with other mechanoreceptors and proprioceptors, they provide continuous sensory feedback during the execution of fine sensorimotor tasks. Functionally, they also detect microscopic slips of objects held within the hand (Johansson & Westling, 1984; Johnson, Yoshioka, & Vega-Bermudez, 2000; Macefield, Hager-Ross, & Johansson, 1996; Srinivasan, Whitehouse, & LaMotte, 1990), which trigger reflexive hand adjustments necessary for effective grip control and precise manipulation of objects (Johansson & Westling, 1984; Macefield et al., 1996; Martin, 1990; Purves, 2008).

Despite the importance of haptic senses to humans and nonhuman primates, surprisingly little is known about the distribution of MCs across digits and species, in either the normal or disease/injury state. In this study we begin to address this by asking three key questions: First, are MCs distributed equally across all digits in macaque monkeys, or does their density more closely reflect digit use? That is, are there more MCs in the skin of opposing digits used in precision grip (digits 1–3, or thumb, index, and middle fingers), versus digits four and five (ring and pinky fingers)? Second, what happens to these peripheral receptors 4–5 months following a spinal cord injury that permanently disrupts their normal innervation, when functional recovery is largely complete? Third, what happens chronically, 12–14 months later?

Previous clinical (Caruso et al., 1994; Herrmann, Boger, Jansen, & Alessi-Fox, 2007; Johansson & Vallbo, 1979; Kelly, Terenghi, Hazari, & Wiberg, 2005) and nonhuman primate (Winkelmann, 1962) studies have reported that MCs are more abundant in the fingertips than the proximal phalanges or palm, which suggests that they concentrate in areas with the greatest tactile spatial acuity and touch sensitivity (Caruso et al., 1994; Dillon, Haynes, & Henneberg, 2001). However, little is known about the relative distribution of MCs across individual digits. One study (Dillon et al., 2001) compared MC density on the index and ring fingers in humans and another compared MC density between digits one, two, and four in numerous primate species, including humans (Verendeev et al., 2015). While both groups reported no differences in MC density across digits, their sampling and statistical methodologies were inconsistent and difficult to interpret.

In addition, little is known about the changes to MCs following spinal injuries that disrupt peripheral innervation to the hand. In humans, injury to the cervical dorsal roots (Anand & Birch, 2002; Berman, Birch, & Anand, 1998; Nagano, 1998), dorsal column (Nathan et al., 1986), or cervical spinal cord (Thomas & Westling, 1995) can cause severe sensorimotor disturbances. Along with impairments in proprioception, patients have difficulty discriminating objects, identifying stimuli moving across the skin, and using their hands in a coordinated manner. As a result, manipulative tasks that require continuous sensory feedback, such as tying shoelaces or buttoning a shirt, become difficult. While there is some clinical evidence to suggest that sensory and motor nerve fibers retain function below the elbow >1 year following cervical SCI (Thomas & Westling, 1995), the state of the peripheral receptors that these fibers once innervated is not clear.

Similarly, cervical dorsal root (C. Darian-Smith, 2007; C. Darian-Smith & Brown, 2000; C. Darian-Smith & Ciferri, 2005; Vierck, 1982) and dorsal column (Cooper, Glendinning, & Vierck, 1993; FERRARO & BARRERA, 1934; Gilman & Denny-Brown, 1966; Glendinning et al., 1992; Leonard, Glendinning, Wilfong, Cooper, & Vierck, 1992; H.-X. Qi et al., 2013; H. X. Qi, Kaas, & Reed, 2014) lesions in monkeys can cause severe cutaneous sensory loss, and a corresponding impairment of digit and hand function. Even so, as long as there is some (<5%) sparing of primary afferent input from the hand, tactile function will return to these digits in the weeks and months following injury. This is evident behaviorally (C. Darian-Smith & Ciferri, 2005), as well as electrophysiologically (C. Darian-Smith, 2004), in the receptive field maps obtained. Following a dorsal root lesion, spared primary afferent axons sprout within the spinal dorsal horn and cuneate nucleus (C. Darian-Smith, 2004), presumably to form new functional connections supporting the recovery of hand function. While we are beginning to understand some of the changes that occur within the CNS following such spinal injuries, it remains unclear what happens to the cutaneous receptors of the initially deafferented digits. Since hand function requires intact sensory processing at all levels, understanding peripheral receptor responses is crucial for developing effective therapeutic strategies to optimize recovery after injury.

In this study, we first characterized the distribution of MCs in all five digits of uninjured macaque hands, which have a similar structure to humans including a fully opposable thumb. We then quantified changes to receptor densities in monkeys that received either a dorsal column lesion (DCL) or a combined dorsal root/dorsal column lesion (DRL/DCL), 4–5 months earlier. Finally, we analyzed the chronic changes 12–14 months later in DRL/DCL-lesioned monkeys. Our findings reveal a unique distribution pattern of MCs across the digits that reflects a close functional relationship between MCs and precision grip. At 4–5 months post-injury, we found a loss of MCs in the fingertips of affected digits following a partial central (DCL) and more complete (DRL/DCL) deafferentation injury. However, 12–14 months following a DRL/DCL, receptor densities in the deafferented digits had returned to normal levels.

Materials and Methods

Animals used

Eleven male macaque monkeys (Macaca fascicularis) were used in this investigation. Monkeys were between the ages of 3–5 years old, with an average weight of 4.1 ± 0.9 kg (as weighed at completion of study). All were colony bred and housed individually at the Stanford Research Animal Facility in adapted four-unit cages (each unit measuring 64 × 60 × 77 cm, depth × width × height, with one monkey occupying all four units). Monkeys were kept to a 12 hour light cycle, received daily enrichment (e.g. novel foods, puzzles, mirrors, videos, and music), with food and water provided ad libitum. Monkeys were assigned to treatment groups randomly, and experimental procedures were in compliance with ARRIVE guidelines. All monkeys were used in other studies, so no new animals were required in the present study. All procedures were conducted in accordance with National Institutes of Health guidelines and were approved by the Stanford University Institutional Animal Care and Use Committee. See Table 1 for details of animals used and the timeline of procedures.

Table 1.

Details of animals used and timing of procedures

Monkey ID	Weight (kg)	Lesion	Weeks between laminectomy and craniotomy	Weeks survived post-lesion
M1601	3.60	Control	n/a	n/a
M1702	2.96	Control	n/a	n/a
M1401	3.60	DCL	11	17.5
M1403	3.52	DCL	13	19
M1402	3.80	DRL/DCL	12.5	19
M1602	4.10	DRL/DCL	12	18
M1603	3.54	DRL/DCL	14	20
M1604	3.94	DRL/DCL	12	18
M1703	5.32	DRL/DCL	46	52
M1704	5.02	DRL/DCL	52	58.5
M1705	6.02	DRL/DCL	51	57

DCL = dorsal column lesion; DRL/DCL = combined dorsal root/dorsal column lesion

Experimental sequence

Monkeys were divided into four groups. Two animals had no lesions and served as controls, two received a dorsal column lesion (DCL) alone and survived for 4–5 months, four received a combined dorsal root/dorsal column lesion (DRL/DCL) and also survived for 4–5 months, and three received a DRL/DCL and survived for 12–14 months.

All lesions were unilateral, and made on the side of the dominant hand (see Fisher et al., 2018). Hand preference was determined over 2–3 weeks prior to the first surgery (laminectomy). Monkeys were handed grapes or other small food items and the preferred hand used to reach and grasp the food was scored over dozens of reaches during this time period.

To make the lesions, a laminectomy was used to expose the C5–C8 region of the cervical cord receiving sensory input from the first three digits (D1–D3) of one hand. Following the initial lesion, monkeys recovered for either 11–14 weeks or 46–52 weeks (Table 1). At this point, they underwent a unilateral craniotomy. This allowed reorganized somatosensory cortex to be identified electrophysiologically for the purposes of injecting axonal tracers to visualize corticospinal projections, as described elsewhere (C. Darian-Smith et al., 2014; Fisher et al., 2018). Monkeys were then kept for an additional ~6 weeks for tracer uptake, before being euthanized.

Surgical procedures

For all surgical procedures, anesthesia was induced using ketamine hydrochloride (10 mg/kg) and maintained throughout surgery with gaseous isoflurane (1–1.5%)/O₂ using an open circuit anesthetic machine. Atropine sulfate (0.05 mg/kg), buprenorphine (0.02 mg/kg), and cefazolin (20 mg/kg) were also administered prior to surgery. An intravenous infusion of Normosol-R was delivered throughout the surgery to preserve fluid balance, and a thermostatically controlled heating pad and air blanket were used to maintain body temperature. Physiological signs were monitored continuously to ensure a proper depth of anesthesia (i.e. heart rate, respiration, blood pressure, pulse oximetry, capnography, and core temperature).

Dorsal root and dorsal column lesions

A laminectomy was made to expose cervical spinal segments C5–C8 (Figure 1), and electrophysiological recordings were conducted from dorsal rootlets to create a microdermatome map of the hand (C. Darian-Smith & Brown, 2000). To produce the dorsal root lesion, rootlets innervating D1–D3 were cut using iridectomy scissors to excise 2–3 mm of each rootlet along the length of the lesion. To produce the DCL, the cuneate fasciculus was cut at the rostral border of detectable input from the thumb using a micro scalpel (Micro-Scalpel, Feather, 15⁰), and the overlying tissues and skin sutured closed. Both procedures were combined to produce DRL/DCLs.

Figure 1.

Photomicrographs showing lesion placement and extent in the nine lesioned monkeys used in this study. (a-g) Transverse sections show the dorsal column lesion core. Green dotted lines delineate the lesion extent, which was variable in size but partial in all animals and only involved the cuneate fasciculus. Sections were background stained with diaminobenzidine (DAB). Adjacent photographs of the spinal cord show the location of the dorsal column lesion (horizontal green bar). (c-g) Adjacent photographs of the spinal cord show the rostrocaudal extent of the dorsal root lesions (vertical yellow bar) in monkeys with combined dorsal root/dorsal column lesions. Scale bar in (a) for (a-f) transverse sections = 1 mm.

Cefazolin (20 mg/kg) and buprenorphine (0.02 mg/kg) were administered to provide a postoperative antibiotic and analgesic, respectively, before the monkeys were returned to their cages. Animals were monitored closely until awake and alert, usually within one hour. No postoperative sequelae were observed in any of the monkeys. Oral buprenorphine (0.015 mg/kg) was administered twice daily for two additional days and meloxicam (0.1 mg/kg) for three to five days post-surgery, as needed.

Craniotomy

At either 11–14 or 46–52 weeks following the initial surgery, all monkeys underwent a unilateral craniotomy (see Table 1). The region of D1–D3 representation was assessed in the primary somatosensory cortex, and a series of anterograde tracers were injected into this region and the corresponding primary motor cortex. Post-operative anesthesia and analgesics were given as described above. The specific details of this procedure can be referenced in previous investigations (C. Darian-Smith et al., 2013; C. Darian-Smith et al., 2014), as they did not form part of the present study.

Electrophysiological recordings

Recordings were made from dorsal rootlets of the cervical spinal cord to identify those with receptive fields on D1–D3. A tungsten microelectrode (1.2–1.4 mΩ at 1 kHz; FHC) was lowered vertically to record from each rootlet, and single or small multiunit extracellular recordings were made from axons within each fascicle to create a microdermatome map. Cutaneous receptive fields were mapped using hand manipulation, a camel hair brush, and Von Frey hairs. Receptive fields were classified as cutaneous if a stimulus force of ≤2.0 g evoked a response. For stimulus forces of >2.0 g, or where joint manipulation or hand movements were required to evoke a response, the receptive field was considered deep. When it was uncertain whether a receptive field was cutaneous or deep, it was classified as cutaneous for the purposes of creating the lesion boundaries. All receptive fields were labeled on hand/body image score sheets and rootlets with cutaneous receptive fields on D1–D3 were cut. Recordings were also made in the primary somatosensory cortex to identify cutaneous receptive fields corresponding to hand representation. Details can be referenced in previous published work (C. Darian-Smith, 2004; C. Darian-Smith & Brown, 2000; C. Darian-Smith & Ciferri, 2005; C. Darian-Smith et al., 2013; C. Darian-Smith et al., 2014).

Perfusion and tissue processing

At the termination of each experiment, monkeys were deeply anesthetized and administered a lethal intravenous dose of sodium pentobarbital (171.6 mg/kg). Animals were then perfused transcardially with 0.1 M phosphate-buffered saline (PBS; pH 7.4) followed by 4% paraformaldehyde. The hands were removed and stored in 4% paraformaldehyde at 4°C until tissue was processed. The brain and spinal cord were also removed for the purposes of other additional studies (C. Darian-Smith et al., 2013; C. Darian-Smith et al., 2014).

Histological preparation of digit pads

A 5 mm diameter full-thickness cutaneous biopsy punch was used to remove samples from the palmar surface of the distal pads of each digit in each animal (Figure 2). Once the tissue was excised and marked at its distal edge (Medical Marker) to maintain orientation, it was cut in half vertically to produce lateral and medial halves. The medial halves were stored in 4% paraformaldehyde for future use. The lateral halves were immersed in 70% ethanol before being embedded on edge in paraffin wax. For each digit, a total of eight transverse sections (4 μm thick) were collected every 100 μm, starting at the middle of the biopsy bunch and extending laterally through the block. These sections were then mounted onto microscope slides and stained with Hematoxylin and Eosin (H&E).

Figure 2.

Photomicrographs of Meissner’s corpuscles. (a) The location of biopsy punches in all monkeys. (b) An example of a transverse section through the skin, showing Meissner’s corpuscles within the dermal papillae, from digit 1 of the lesioned hand of monkey M1402. (c) Examples of individual Meissner’s corpuscles from targeted (D1–D3) and non-targeted (D4–D5) digits of the ipsilateral hands of monkeys within each treatment group. Meissner’s corpuscles are imaged from digit 1 and digit 5 of DCL-lesioned monkey M1403, digit 1 and digit 4 of short-term DRL/DCL-lesioned monkey M1602, and digit 2 and digit 4 of long-term DRL/DCL-lesioned monkey M1704. No differences in Meissner’s corpuscle morphology were observed across digit, hand, or treatment group using H&E staining at the light microscopy level. Scale bars = 50 μm.

Microscopy and data collection

Meissner’s corpuscles were identified (Figure 2) and quantified using a Zeiss Axiophot light microscope (Carl Zeiss Microscopy, LLC, Thornwood, NY, USA) coupled to AxioVision image processing software (AxioVision, RRID:SCR_002677). For counting, prospective MCs were classified based on three criteria: 1) the receptor was located within the dermal papillae, 2) the long axis was perpendicular to the skin surface, and 3) the arrangement of lamellar cells was parallel to the skin surface. We did not include structures that did not meet all three criteria, so any MCs that may have atrophied and lost their structural integrity, were unrecognizable as MCs and were not included. Counts were made and verified by two different investigators. The first knew the identity of the animal but was blind to the hand or digit, and the second investigator was completely blind to the animal, hand, and digit being examined. Counts between investigators were identical. Following the counting process, measurements were made of the cross-sectional diameter of MCs and the length of each sample along the stratum granulosum (Figure 2).

Statistical analysis

Three statistical analyses were conducted – one to assess MC distributions in uninjured hands, one to assess changes following each of two different primary afferent spinal lesions at 4–5 months following injury, and one to assess chronic changes in DRL/DCL monkeys at 12–14 months following injury. Data were analyzed using general linear mixed models (GLMM) in the statistical software JMP 13.1.0. All models included repeated measures, and so monkey was entered as a random effect.

The first model was constructed to analyze the distribution of receptor density across digits in uninjured hands. Data were included from both hands of control monkeys and from contralateral hands of lesioned monkeys, in a model that included digit as a categorical fixed effect, and its interaction with monkey. The outcome of interest was the density of Meissner’s corpuscles (receptors per mm²), averaged across eight biopsy sections per digit. Density was estimated using Abercrombie’s formula (Abercrombie, 1946; Verendeev et al., 2015) plus an additional correction factor, which is used to transform a measure of “receptors per section” into an estimate of “receptors per mm²”. This formula is given by: N = n*(T/T+H), where n is the average number of receptors per section, T is the average thickness of each section, H is the average cross-sectional diameter of the MCs, and N is the corrected average number of receptors per section for each digit. This value is then transformed into an estimate of density (receptors per mm²) by dividing by the average area of epidermis measured for each digit calculated across the 8 sections. We used the same mean cross-sectional diameter (25.1 μm) across the entire dataset, which was derived from measuring all receptors from one biopsy section for each digit of each monkey, for a total of 534 receptors. Cross-sectional diameters were measured from the fourth tissue section of each biopsy punch.

The next two models were constructed to analyze the effects of lesions on receptor density. In both models, we used as our dependent variable, the logarithm of the ratio of receptor densities in corresponding ipsilateral and contralateral digits. The logarithmic transformation is advantageous as it tends to normalize distributions of ratios in situations where the numerator and denominator have similar values. Because the logarithm of one is zero, logarithmic values above zero represent cases where ipsilateral receptor densities are greater than matched contralateral values, and vice versa for values below zero.

Because lesions were made to selectively affect D1–D3 while sparing D4–D5, we created corresponding “targeted” and “non-targeted” groups in our analysis of lesion effects on receptor density. We constructed models that included treatment (i.e. control, DCL, or short-term DRL/DCL in the first model; control, short-term DRL/DCL, or long-term DRL/DCL in the second model), digit group (i.e. targeted or non-targeted), and digit as categorical fixed effects. Monkey was nested within treatment and digit within digit group. These models included all possible interactions and were the final versions used.

All tests were two-tailed at alpha = 0.05. Significant fixed effects in both models were followed up with Bonferroni-corrected post hoc tests. Model assumptions (normality of error, homogeneity of variance, and linearity) were verified by visual inspection of diagnostic plots. All values are reported as least square means ± standard error.

Results

Statistical analyses were performed on data collected from 11 monkeys and 110 digits (2 control, 2 DCL, 4 short-term DRL/DCL, and 3 long-term DRL/DCL; Table 1). Lesions were unilateral, and did not affect the contralateral hand. Four digits (and the ratios that involved these digits) were excluded from the analyses due to physiological abnormalities (i.e. samples had no dermal papillae). These were the contralateral digit 4 of a DCL-lesioned monkey, and contralateral digits 1–3 of a control monkey.

Figure 1 shows the extent of lesions in the DCL, short-term DRL/DCL, and long-term DRL/DCL animals. The dorsal column lesion was variable in size but partial in all animals and only involved the cuneate fasciculus. Figure 2 shows the typical appearance and location of Meissner’s corpuscles within the dermal papillae, in H&E stained tissue at the light microscopy level. Additional images (Figure 2c) are included to show comparisons between MC morphology in targeted and non-targeted digits of monkeys within each treatment group. The location of the biopsy punches is also shown (Figure 2a), and was the same for all monkeys.

MC distributions in uninjured hands

To assess the distribution patterns of MCs across the digits, 13 hands, including 4 from control monkeys and 9 (contralateral) from lesioned animals, were used. As illustrated in Figure 3, receptor densities were found to be significantly different between digits (F_4,38.6 = 34.24, p < .0001), such that the greatest density of MCs was observed on the thumb (43.6 ± 1.8 receptors/mm²). The index finger averaged 35.3 ± 1.8 receptors/mm², and digits 3–5 had significantly lower densities (27.0 ± 1.8, 27.8 ± 1.9, and 28.5 ± 1.8 receptors/mm², respectively). Bonferroni-corrected post hoc tests showed that receptor density was significantly higher in digit 1 than in all others, and significantly higher in digit 2 than in digits 3–5 (Figure 3). These findings showed a clear distinction between the opposing thumb and index finger, and the middle, ring, and pinky fingers which are not used in the same way for fine precision grip behaviors (e.g. grooming, foraging, etc.).

Figure 3.

Comparison of Meissner’s corpuscle receptor densities between individual digits of uninjured hands. Meissner’s corpuscle receptor densities were significantly higher in digit one than in all other digits, and significantly higher in digit two than in digits three through five. Uninjured hands included the contralateral hands of lesioned monkeys and both hands of nonlesioned controls. Significant differences are indicated by an asterisk (*). Values displayed are least square means ± standard error.

MC distributions at 4–5 months following primary afferent lesions – i.e. DCLs and DRL/DCLs

In our second analysis, we asked what happens to the MC densities following either a DCL or a combined DRL/DCL at 4–5 months following injury. Ipsilateral to contralateral receptor density ratios were affected by a significant interaction between digit group (i.e. targeted = D1–D3 and non-targeted = D4–D5) and treatment (i.e. control, DCL, and short-term DRL/DCL; F_2,12.1 = 7.66, p = .007).

Bonferroni-corrected post hoc tests within each combination of treatment and digit group showed that densities were significantly lower on the ipsilateral side compared to the corresponding contralateral side in just the targeted digits of short-term DRL/DCL-lesioned monkeys (shown by a logarithmic value below zero, which represents an ipsilateral-to-contralateral ratio below 1; Figure 4).

Figure 4.

Ipsilateral to contralateral receptor density ratios within each combination of treatment (control, DCL and short-term DRL/DCL) and digit group (targeted and non-targeted). Meissner’s corpuscle receptor densities were significantly lower on the ipsilateral hand as compared to the corresponding contralateral hand in the targeted digits of short-term DRL/DCL-lesioned monkeys. Significant differences between ipsilateral and contralateral densities (ratios different from one) are indicated by an asterisk (*). Values displayed are back-transformed least square means ± standard error.

Bonferroni-corrected post hoc tests were used to compare treatments within each digit group, and Figure 5 illustrates this. These data showed that short-term DRL/DCL-lesioned monkeys had significantly lower ipsilateral to contralateral ratios in the targeted digits than both DCL-lesioned and control monkeys, suggesting a stronger effect of the lesion.

Figure 5.

Ipsilateral to contralateral receptor density ratios for each combination of treatment (control, DCL and short-term DRL/DCL) and digit group (targeted and non-targeted). Within targeted digits, the ipsilateral to contralateral receptor density ratio was significantly lower in short-term DRL/DCL animals as compared to both DCL-lesioned and control monkeys. Significant differences between treatments are indicated by an asterisk (*). Values displayed are back-transformed least square means ± standard error.

There was no significant main effect of digit (F_3,15.0 = 0.43, p = .736), meaning that targeted digits D1–D3 were all affected in the same way, and non-targeted digits D4–D5 were also affected similarly to each other, so lesion effects appeared to be more or less uniform within digit groups (targeted and non-targeted).

MC distributions at 12–14 months following a combined DRL/DCL

In our third analysis, we asked whether MC densities measured 12–14 months after a DRL/DCL (long-term) differed from those measured 4–5 months after a DRL/DCL (short-term) and from controls. Receptor densities were affected by a significant interaction between digit group (i.e. targeted and non-targeted) and treatment (i.e. control, short-term DRL/DCL, and long-term DRL/DCL; F_2,13.5 = 13.71, p = .0006).

Bonferroni-corrected post hoc tests within each combination of treatment and digit group showed that ipsilateral receptor densities were significantly lower than contralateral receptor densities in just the targeted digits of short-term DRL/DCL-lesioned monkeys. There was no such difference in long-term DRL/DCL-lesioned monkeys (Figure 6).

Figure 6.

Ipsilateral to contralateral receptor density ratios within each combination of treatment (control, short-term DRL/DCL, and long-term DRL/DCL) and digit group (targeted and non-targeted). Ipsilateral receptor densities were significantly lower than contralateral receptor densities in the targeted digits of short-term DRL/DCL-lesioned monkeys, but no such difference was observed in long-term DRL/DCL-lesioned monkeys. Significant differences between ipsilateral and contralateral densities (ratios different from one) are indicated by an asterisk (*). Values displayed are back-transformed least square means ± standard error.

Bonferroni-corrected post hoc tests were used to compare treatments within each digit group, and Figure 7 illustrates this. These data showed that short-term DRL/DCL-lesioned monkeys had significantly lower ipsilateral to contralateral receptor density ratios in targeted digits than was observed in the same digits of control and long-term DRL/DCL-lesioned monkeys. The long-term DRL/DCL-lesioned monkeys did not significantly differ from the control monkeys in either digit group.

Figure 7.

Ipsilateral to contralateral receptor density ratios for each combination of treatment (control, short-term DRL/DCL, and long-term DRL/DCL) and digit group (targeted and non-targeted). Short-term DRL/DCL-lesioned monkeys had significantly lower ipsilateral to contralateral receptor density ratios in targeted digits as compared to the same digits in control and long-term DRL/DCL-lesioned monkeys, whereas long-term DRL/DCL-lesioned monkeys did not significantly differ from the control monkeys. Significant differences between treatments are indicated by an asterisk (*). Values displayed are back-transformed least square means ± standard error.

Again, there was no significant main effect of digit (F_3,19.5 = 0.23, p = .872), meaning that targeted digits D1–D3 were all affected in the same way, and digits 4 and 5 were also affected similarly to each other, so lesion effects appeared to be more or less uniform within digit groups (targeted and non-targeted).

Summary

In summary, in normal macaque monkeys, MCs are most concentrated on the distal pad of the thumb and index fingers, compared with the third through fifth digits (Figure 3). Greatest densities are found on the thumb (43.6 ± 1.8 receptors/mm²) followed by the index finger (35.3 ± 1.8 receptors/mm²), and the remaining digits D3–D5 had similar densities (27.0 ± 1.8, 27.8 ± 1.9, and 28.5 ± 1.8 receptors/mm², respectively) that were less than in the opposing digits.

Four to five months following a partial central (DCL) or more complete (DRL/DCL) deafferentation, there was a loss of MCs on the distal glabrous pads of each of the affected digits (Figure 4). Though the loss of MCs was observed in targeted digits in both DCL and DRL/DCL monkeys, the loss was only significant in DRL/DCL monkeys (a reduction from 32.8 ± 2.1 receptors/mm² in uninjured hands to 23.8 ± 2.1 receptors/mm² in injured hands, which represents a ~30% reduction in receptor density), presumably due to the smaller DCL sample size and less complete deafferentation.

Twelve to fourteen months following a DRL/DCL, the density of MCs in the targeted digits of injured hands had returned to normal levels (Figures 6 and 7). This occurred despite a permanent loss of original afferent innervation to the MC receptor population.

Discussion

To understand how Meissner’s corpuscles are involved in restoring sensory input following cervical spinal cord injury, we sought to answer three major questions. First, what is the distribution pattern of MCs in the distal pads of all five digits in the macaque monkey, which has a similar hand structure to humans and a fully opposable thumb? Second, are there any quantitative changes to the density of these mechanoreceptors 4–5 months after animals received either a DCL or a combined DRL/DCL? Finally, what happens chronically at 12–14 months following the initial deafferentation injury?

MC distributions in normal digits

Our findings in normal hands show that MCs are more densely distributed in the distal pad of the thumb and index fingers than in digits three through five, with the greatest concentration on the thumb. This is a distribution pattern that reflects oppositional digit use, and it suggests that humans may also show differences in receptor densities across digits.

Of the few studies that have compared mechanoreceptor densities across multiple digits, all use different methodologies and most focus on comparative analyses between different primate species (Bolanowski & Pawson, 2003; Guclu, Bolanowski, & Pawson, 2003; Winkelmann, 1963). To address previous inconsistencies, Verendeev and colleagues (2015) attempted a more systematic analysis in different primate species, but found no statistical differences in MC density between digits one, two, and four or between hands of any given species. Though their conclusion runs counter to the findings of the present study, the disparity is almost certainly methodological. In the Verendeev study, samples were taken from mostly older subjects (i.e. 78–91 years for humans; three of four chimpanzees were 44–57 years; macaques were 13 and 20 years old), and it is known that MC density and tactile acuity decline markedly with age (Bolton, Winkelmann, & Dyck, 1966; Dickens, Winkelmann, & Mulder, 1963; Iwasaki, Goto, Goto, Ezure, & Moriyama, 2003). In addition, only three to four sections of unspecified length or location on the digit pads were analyzed per digit, and data was pooled across subjects. In the present study, all samples were from eleven young adult animals, and eight sections of ~5 mm length and comparable pad location were analyzed per digit (i.e. biopsy sample), using multi-factorial statistical analyses to determine significance. Importantly, our statistical modeling allowed for digit comparisons within individuals, which normalized for density variations observed between individuals.

Our findings, in contrast to earlier reports, indicate that there is a close functional relationship between MC density and precision grip. Precision grip involves the opposition of the thumb with another digit, usually the index finger, to manipulate items and perform volitional tasks (Landsmeer, 1962; Napier, 1956). This coincides with our findings in normal hands in which the density of MCs is greater in the thumb and index finger than in digits three through five (Figure 3). Meissner’s corpuscles also help detect slips of objects within the hand, which enable compensatory changes in grip control and improved dexterity (Johansson & Westling, 1984; Johnson et al., 2000; Macefield et al., 1996; Martin, 1990; Purves, 2008; Srinivasan et al., 1990). It is not surprising then that the two digits used most often in fine manipulative tasks contain the greatest concentration of MCs. The significantly greater number of MCs on the thumb versus the index finger underscores the importance of the thumb in all oppositional tasks.

Additional support for the relationship between MC density and precision grip comes from sensory tests that measure tactile spatial acuity, namely the moving two-point discrimination test which characterizes the rapidly adapting receptor system (i.e. MCs). Of the two studies that expressly used this technique (Louis et al., 1984; Vega–Bermudez & Johnson, 2001), both observed that spatial acuity declines from the index to the ring finger; an observation that highlights the importance of the index finger in complex tactile and oppositional movements.

If MC densities correlate directly with digit use, as is suggested by our findings, would we expect to see a similar distribution pattern across the fingertip pads of the human hand? In short, the answer is unclear. Though strikingly similar in many ways, there are notable differences between human and macaque hand structure and function. Structurally, humans have one more muscle in their hand than macaques and the human thumb is long relative to the other digits, while in macaques the thumb is comparatively short. Functionally, only humans can oppose their thumb with the distal pad of any of the other digits. In addition, only humans have ulnar opposition, which allows the ulnar side of the hand to flex towards the base of the thumb. So, while it is true that humans, like macaques, use thumb-index opposition for the very finest of pincer grips, it is also true that humans have greater oppositional versatility than any other extant primate species (including apes) and can oppose the thumb independently with each of digits two through five. This behavioral difference may affect receptor density distribution patterns, and requires further study. Importantly, while a number of studies have compared two digits, or compared single digits with palmar skin, none of these investigations have adequately compared MC densities across all five digits in individual human subjects (i.e. whereby subjects were of comparable age and health, biopsies were taken from similar locations, and where multifactorial statistical analyses were used), so clinical observations remain confusing and incomplete.

MC distribution pattern changes at 4–5 months following spinal deafferentation injuries

In monkeys that had a primary afferent spinal lesion, our data show that there is a significant loss of MCs on just the digits deafferented by the lesion in the next 4–5 months following the injury. While the trend was similar for the two lesions, the greatest loss of MCs occurred on the targeted digits of monkeys that received a combined DRL/DCL. Reasons for the lesion differences are considered below, but simply put, the DRL component of the combined lesion produces a more complete deafferentation than the DCL alone (which leaves spinothalamic input intact).

Following the specific lesions examined in this study, there was a permanent loss of primary afferent innervation to most of the MCs within the skin of the first three digits. The morphological and biochemical integrity of MCs depend on intact axonal input (Ide, 1982; Marquez, Perez-Perez, Naves, & Vega, 1997; Munger & Ide, 1988), and so presumably, the receptors that are removed from the skin, or which degenerate (Dellon, 1976), are those that lose most or all of their original innervation. Importantly, the significant loss of MCs occurred in the skin during the time that we see the greatest behavioral recovery (C. Darian-Smith & Ciferri, 2005; unpublished observations, 2018). This suggests that for the return of any tactile sensitivity to the digits during this time period, the reduced MC receptor populations in D1–D3 of the injured hand (which comprise ~70% of the concentration of MCs on the uninjured contralateral hand) must be receiving innervation from relatively few spared primary afferent neurons. So, even though the active primary afferent population is permanently and extensively depleted by the lesions (to <5% of the original population), peripheral axon innervation to the remaining receptors is demonstrably sufficient to facilitate a functionally useful signal to the spinal cord and all higher levels. Whether or not this process (at this time point) involves terminal sprouting of these functionally relevant terminals (or the strengthening of pre-existing connections), remains unclear because human evidence suggests that a single sensory neuron can branch to innervate a large number of MCs (i.e. 14–28 MCs in human digits; Johansson, 1978). In addition, each MC is innervated by more than one cutaneous axon (i.e. 2–9 fibers supplying a single receptor; Cauna, 1956), though it is not known how many neurons these terminals originate from, how these ratios change following injury, or even what is required for functional reinnervation.

MC distribution pattern changes at 12–14 months following deafferentation

Our findings in chronic monkeys show that by 12–14 months post-lesion, there is a remarkable restoration of MC numbers in the skin of the deafferented digits, despite the (~95%) permanent loss of most of their original innervation. This indicates that lost or degenerated receptors are gradually innervated (or re-innervated), and this must occur via the sprouting of terminal fibers from spared neighboring primary afferents. Since most functional recovery has already occurred by the fifth post-lesion month (C. Darian-Smith & Ciferri, 2005), the added benefit of having more receptors by the 12–14 month period is likely to be more subtle. Whether or not MCs are lost completely and repopulate the dermal papillae de novo, or atrophy beyond recognition and await reinnervation to recover morphological structure, is not known, but peripheral nerve transection studies suggest the latter may be more likely. Dellon and colleagues (1975) found that following peripheral nerve transection, axon terminals degenerated within a couple of weeks, and MCs lost their structural integrity by 4 months. Dellon (1976) also reported that 9 months after digital nerve transection and resuture, atrophied MCs could be reinnervated and appeared normal.

In short, mechanoreceptor populations undergo a remarkable, albeit poorly understood, reorganization at the periphery, which informs and necessarily shapes all of the central changes that are known to occur throughout the neuraxis during the recovery process. Some of these central changes have even been reported in the same monkeys used in this study (C. Darian-Smith et al., 2014; Fisher et al., 2018). It should be noted that the lesions used in the present study were purposely small (Figure 1), and strictly localized to the first three digits. Even so, our observations can be extended conceptually to any clinical spinal deafferentation injury.

MC innervation

At a cellular level, MCs are surprisingly complex. In addition to being specialized for mechanosensation and innervated by large myelinated Aαβ fibers, it has been known for nearly two decades that they also receive some direct innervation from C fibers (Pare, Elde, Mazurkiewicz, Smith, & Rice, 2001), which are associated with the transmission of pain. As a consequence, MCs may play an important role in the perception of both texture and mechanically induced pain (e.g. allodynia). Chronic pain affects ~two thirds of SCI patients (one third reporting it to be severe; Siddall, McClelland, Rutkowski, & Cousins, 2003), but is poorly understood and few studies have pursued the underlying cause, in part because nonhuman primates mask symptoms to avoid negative social repercussions. C fibers are known to be able to sprout profusely at the site of a peripheral injury (Brenan, 1986; Doucette & Diamond, 1987; Pertovaara, 1988), so their innervation of MCs following deafferentation injuries may contribute to the clinical presentation of pain. That is, the relative weighting of C and Aαβ fiber innervation to MCs may be altered in a maladaptive way following injury. In the present study, a less dramatic loss of MCs in monkeys receiving the DCL on its own is likely due to the fact that C fiber innervation was almost completely intact in these animals.

Importantly, our data show that an initial reduction of receptors (~30% loss) at 4–5 months post-lesion gives rise to normal levels in chronic monkeys surviving post-lesion for >1 year. This is intriguing and raises additional questions: Are the restored receptors rescued MCs that initially atrophied (and remained undetected) in the dermal papilla, or do they develop de novo in response to changes in terminal innervation patterns? Presumably they are reinnervated by the sprouting of spared primary afferent fibers that already innervate MCs in neighboring skin, but is this the case? Given that a greatly reduced fiber population is available to innervate many more receptors at the periphery than normal (Johansson, 1978), does this lead to a permanently reduced sensitivity (i.e. larger receptive fields)? Finally, the present study shows changes in just one receptor population, but are there similar changes in other populations (e.g. Merkel cells, Pacinian corpuscles, Ruffini endings, proprioceptors, etc.)? Since peripheral sensation is key to all higher level reorganization and recovery, further studies are needed to address these questions so that therapeutic clinical strategies can be developed that optimize receptor regeneration and reinnervation following spinal cord injury.

Figure 8.

Schematic summary showing changes in peripheral receptor density and innervation at 4–5 and 12–14 months following a combined dorsal root/dorsal column lesion. The lesioned primary afferents are permanently lost, so whether or not MCs are completely lost, or just atrophied at 4–5 months, their detectable density returns to normal by one year post-lesion, and these MCs must be innervated by spared primary afferent fibers.