Redistribution of inhibitory force feedback between a long toe flexor and the major ankle extensor muscles following spinal cord injury

Abstract

Inhibitory pathways from Golgi tendon organs project widely between muscles crossing different joints and axes of rotation. Evidence suggests that the strength and distribution of this intermuscular inhibition is dependent on motor task and corresponding signals from the brainstem. The purpose of the present study was to investigate whether this sensory network is altered after spinal cord hemisection as a potential explanation for motor deficits observed after spinal cord injury. Force feedback was assessed between the long toe flexor and ankle plantarflexor (flexor hallucis longus), and the three major ankle extensors, (combined gastrocnemius, soleus and plantaris muscles) in the hind limbs of unanesthetized, decerebrate, female, cats. Data were collected from animals with intact spinal cords (control) and lateral spinal hemisections (LSHs) including chronic LSH (4–20 weeks), sub-chronic LSH (2 weeks) and acute LSH. Muscles were stretched individually and in pairwise combinations to measure intermuscular feedback between the toe flexor and each of the ankle extensors. In control animals three patterns were observed (balanced inhibition between toe flexor and ankle extensors, stronger inhibition from toe flexor to ankle extensor or vice versa). Following spinal hemisection, only strong inhibition from toe flexors onto ankle extensors was observed independent of survival time. The results suggest immediate and permanent reorganization of force feedback in the injured spinal cord. The altered strength and distribution of force feedback after spinal cord injury may be an important future target for rehabilitation.

Graphical Abstract

Golgi tendon organ pathways contribute to coordination of limb extensors during motor tasks including locomotion. Normally, these inhibitory force feedback pathways are flexibly weighted. We show that following spinal cord injury, force feedback is permanently reorganized. Balance is lost and biased inhibition emerges from toe flexors onto ankle extensors.

INTRODUCTION

Spinal cord injury (SCI) results not only in some combination of weakness and paralysis (Steven et al., 2011), but also in dysfunction of neural pathways in the spinal cord, such as spasticity and clasp-knife inhibition (Burns et al., 2012; Ditunno and Formal, 1994; Fawcett et al., 2006, little et al., 1999; Scheel-Sailer et al., 2019). These clinical signs are usually attributed to alterations in neural pathways arising from muscle spindle receptors (Eccles et al. 1957a; Lloyd, 1946; Nichols, 1999; Nichols and Koffler-Smulevitz, 1991; Wilmink and Nichols, 2003) or from group III or group IV receptors (Rymer and Houk, 1979; Cleland and Rymer, 1990) in the case of clasp-knife inhibition. Relatively less is known about the influence of spinal cord injury on pathways arising from Golgi tendon organs, force receptors located at the junction of muscle and tendon or aponeurosis of origin (Houk, 1972; Houk, 1979; Houk and Henneman, 1967; Jansen and Rudjord, 1965).

Tendon organ pathways form a distributed, intermuscular network in the feline spinal cord (Eccles et al., 1957) that links extensor muscles across joints and axes of rotation through inhibitory pathways (Nichols, Bunderson and Lyle, 2016). Excitatory pathways from tendon organs are expressed during locomotion (Pearson and Collins, 1993; Guertin et al., 1995), but co-exist with the inhibitory network (Ross, 2006). In the decerebrate state, the relative weightings of the inhibitory, intermuscular linkages are variable (Bonasera and Nichols, 1994; Lyle and Nichols, 2018). For example, the magnitude of inhibition between two muscles may be approximately balanced, or biased in one direction or the other (Lyle and Nichols, 2018). During spontaneous stepping in premammillary decerebrate animals, one distribution predominates, wherein the inhibition is focused on distal extensor muscles (Ross and Nichols, 2009). This proximal to distal gradient of inhibition may serve to increase the compliance of the distal joints to provide an appropriate mechanical interface with the ground (Nichols, 2018). These observations suggest that the distribution of inhibitory force feedback may vary according to the requirements of different tasks, such as walking, running, jumping and landing (Lyle and Nichols, 2018).

These considerations led us to ask how the distribution of inhibition would be affected by spinal cord injury. It had been suggested that spinal cord injury causes an amplification of inhibitory pathways (Eccles, 1959), but it was not clear from these electrophysiological studies how the intermuscular distribution is affected by the injury or how any alterations in the pattern of force feedback would be related to the motor deficits of animals with spinal cord injury. More recent results suggest that spinal cord injury alters sensory pathways in more complex ways than simple amplification or suppression (Frigon et al., 2008; Rossignol and Frigon, 2011). For example, it was shown that stimulation of cutaneous nerves in animals with chronic spinal hemisections led to increases or decreases in short-latency responses of homologous muscles depending on the side of the lesion (Frigon et al., 2009). In another study, excitatory responses were obtained in the semitendinosus and sartorius muscles following stretch of the triceps surae muscles, but only after spinal cord transection (Frigon et al., 2011). In the case of force feedback, would a variable pattern of inhibition be observed across animals but simply amplified, or would a pattern unique to spinal cord injury be seen? The answer to this question is significant in identifying the mechanisms underlying the observed motor deficits in animals with partial spinal cord injury.

In the research reported here, we studied the strength and distribution of inhibition between the long toe flexor, flexor hallucis longus (FHL), and the major ankle extensors, combined medial and lateral gastrocnemius (G), soleus (SOL) and plantaris (PLANT) muscles. According to our prior studies, the major ankle extensors exchange substantial inhibition with FHL, which in the quiescent decerebrate animal can be relatively balanced in both directions, or biased toward FHL or toward G, SOL or PLANT (Lyle and Nichols, 2018). The lateral spinal cord hemisection (LSH) lesion model was used as we (Mondello et al., 2015; Doperalski et al., 2011; Jefferson et al., 2011; Tester and Howland, 2008) and others (Basso et al., 1994; Eidelberg et al., 1986; Helgren and Goldberger, 1993; Barriere et al., 2008) have shown that general locomotor recovery on level surfaces is relatively quick and substantial in these animals while the major descending supraspinal pathways, including those thought to modulate force feedback (Nichols et al, 2014) are interrupted, primarily on one side. We observed only one force feedback pattern following spinal hemisection, namely, strong inhibition from FHL onto G, SOL and PLANT and little or no inhibition in the opposite direction. In addition, the altered distribution of force feedback was observed bilaterally. Furthermore, we found that this directional bias appeared soon after spinal injury and remained unchanged for several months. Preliminary data have been reported in abstracts (Niazi, 2012, 2014). Behavioral studies of animals with chronic spinal hemisection walking on simple and difficult surfaces (Doperalski et al., 2011; Jefferson et al., 2011; Kuhtz-Buschbeck et al., 1996; Basso et al., 1994) have revealed motor disturbances that are consistent with an increase in inhibition to muscles active during stance (extensors).

METHODS

All procedures were completed in accordance with guidelines from the National Institutes of Health (NIH) and approved by the Institutional Care and Use Committees (IACUC) at Georgia Institute of Technology, the University of Louisville, the Robley Rex VA Medical Center and the University of Florida. The experiments were performed on 14 purpose-bred, adult (>1 year of age), female cats (Liberty Research, Inc, New York). Six of the cats were tested after a chronic spinal injury (see below), 2 after acute spinal injury and 6 otherwise naive/control cats were tested for comparison. Experiments performed after acute and chronic spinal injury (ranging 2 to 20 weeks) allowed for examining the potential effect of time since injury on inhibitory force feedback. The consistent findings (see results, e.g. Figures 7 and 9) led us to limit cat numbers over the time range. The experiments were conducted on both legs in each animal, providing an opportunity to compare the effects of the unilateral lesions on the ipsilesional (ipsilateral) and contralesional (contralateral) limbs. All cats were socially housed. It should be noted that data from 2 of 6 of the control animals also were used in a prior publication (Lyle and Nichols, 2018).

Figure 7.

Comparison of force-dependent inhibition expressed as percent change (state 1 vs state 2) across animals for the late epoch (110–150 ms) and plotted versus background forces (N). (a, b) Shown are sub-chronic (Cat-1: 2 weeks), chronic LSH (Cat-2: 4 weeks, Cat-3: 4 weeks, Cat-4: 4 weeks, Cat-5: 8 weeks, Cat-6: 20 weeks) and (c, d) control animals. Solid lines represent data with G as the recipient, and dashed lines represent FHL as the recipient. The line colors represent different experiments/animals. Note the larger inhibition from FHL onto G (solid) compared to G onto FHL (dotted) bilaterally for the cats with LSH, at p ≤ 0.001, across animals. The inhibition was generally stronger in the ipsilateral limb (a) in comparison to contralateral limb (b). (c, d) Variability in both the magnitude and directional bias of the inhibition between FHL and G across preparations in control animals in both left and right limbs respectively. *FHL, flexor hallucis longus; G, gastrocnemius.

Figure 9.

Quiescent data showing inhibition between G and FHL in animals with chronic LSH. Individual data points are late epoch responses expressed as percent inhibition during trials with constant background forces obtained from animals with chronic LSH. Note the directional bias of inhibition from FHL onto G bilaterally across animals irrespective of the time post LSH. The relative inhibition from FHL onto G was significantly larger than G onto FHL bilaterally (Wilcoxon signed rank test: p=0.04 for both limbs), whereas no difference was found for FHL onto G or G onto FHL between limbs (Wilcoxon signed rank test: p>0.05).

Thoracic hemisection surgical procedure:

Lateral spinal hemisection was performed under general anesthesia and aseptic conditions (Jefferson et al., 2011). Atropine sulfate (.04 mg/kg, SC) and acepromazine (0.4 mg/kg, SC) were given preoperatively. Isoflurane (1–4%, .8–1.2L O₂₎ was used to induce and maintain a surgical plane of anesthesia. Lactated ringers (10 ml/kg/h) was administered for hydration and vitals (respiratory rate, heart rate, expired CO₂, SpO₂, body temperature, EKG) monitored continuously throughout surgery and maintained within normal physiological parameters. Sterile surgical procedures were strictly observed. After a 1.5 to 2” midline incision extending from the T8 to T11 dorsal processes, a laminectomy was performed at T9 and part of T10 and the dura mater carefully incised with a small blade. The LSH was completed with iridectomy scissors between the caudal T9 and rostral T10 spinal levels. The dura was sutured using 8–0 Prolene (Ethicon). Durafilm and then Gelfoam were placed over the dural sutures. The muscle, subcutaneous layer and skin were closed in layers (2–0 Dexon II and 3–0 Dexon II respectively). Buprenorphine (0.02 mg/kg, TID) was given for 48 hours and the bladder monitored for fullness and emptied using the Crede method until voluntary voiding recovered during the first post-operative week. Penicillin G procaine or benzathine (40,000U/kg body weight, IM) was given prophylactically for either three days, the day before, the day of and the day after surgery, or at the time of surgery respectively. After the hemisection, the animals were permanently housed on thick foam cushions which covered the entire cage floor and resting boards removed to prevent peripheral nerve compression, pressure sores, and skin breakdown. The resting energy requirement (Bermingham et al., 2010) of each spinal cord injured animal was calculated and caloric intake monitored to assure proper nutrition and maintenance of a good body condition score (BCS). All animals used in the sub-chronic and chronic SCI groups were spayed females. These procedures are standard in the laboratory, most have been reported in detail previously as well as the rationale for use of spayed females to diminish the impact of hormone levels and challenges associated with bladder care in males (Mondello et al., 2015; Jefferson et al., 2011).

Terminal experiments for animals with sub-chronic 2 weeks (1 cat/ cat-1) or chronic SCI’s for, 4 weeks (3 cats/ cats-2, 3 & 4), 8 weeks (1 cat/ cat-5) and 20 weeks (1 cat/ cat-6) were completed after surgical recovery. In two animals, an acute LSH was made immediately after collection of control force feedback data. For these acute injuries, the decerebrate preparation was temporarily placed back under isoflurane anesthesia (no other drug), the spinal cord exposed and a lateral hemisection made with iridectomy scissors as described above. Isoflurane anesthesia then was titrated down over 30 minutes and discontinued, and the injury site covered with saline soaked Gelfoam and gauze for the remainder of the data collection during the terminal experiment.

Surgical procedures for terminal reflex experiment:

The procedures used for evaluating intermuscular reflex circuitry have been reported previously in detail (Nichols, 1994; Ross and Nichols, 2009; Lyle and Nichols, 2018). After a surgical plane of isoflurane gas anesthesia was achieved, tracheal intubation was performed and 1–3% isoflurane used thereafter to maintain deep anesthesia. A cannula was inserted into an external jugular vein to administer saline during the experiment. Adequate anesthesia was confirmed during the experiment by absence of withdrawal reflexes. Heart rate, respiratory rate, oxygen saturation, expired carbon dioxide, and core body temperature were monitored during all procedures. A heating pad was used to maintain core body temperature at 37° C.

The head was fixed in a stereotaxic frame, the abdomen was supported by a sling, and the hindlimbs were rigidly fixed. Hindlimb fixation with the knee at 110° angle was achieved using intramedullary rods into the femur and tibia that were clamped together and rigidly fixed to the support frame. The distal tibia was rigidly fixed at 90° angle with an ankle clamp on the malleoli and attached to the support frame.

The gastrocnemius muscle, with both heads attached (G), flexor hallucis longus (FHL), soleus (SOL) and plantaris (PLANT) muscles were dissected in both the right and left hindlimbs. Muscles were carefully separated free from adjacent tissues and muscles to minimize mechanical coupling while being careful to preserve their nerve and vascular supply. The tendon of FHL was cut where it merges with the flexor digitorum longus tendon. The tendons of G, SOL and PLANT were carefully separated at their common insertion onto the calcaneus. The tendon of PLANT was released from its connection to the calcaneus, and cut distally near its insertion to the flexor digitorum brevis muscle. G and SOL were released distally at their calcaneal insertion with a small bone chip. SOL and PLANT were then carefully separated from the G proximally. The tendons were attached to custom tendon clamps. The tendon clamps were connected to strain gauge myographs in series with linear motors. Experimental setup is shown in Figure 1.

Figure 1.

Experimental setup. Muscles of both limbs were connected in pairs to servo-controlled linear motors by way of strain gauge myographs. Bones of the hind limbs were immobilized using pins inserted longitudinally into the femur and tibia at the knee, and ankle clamps at the medial and lateral malleoli, while maintaining the knee at 110° angle and ankle at 90° angle (mid-stance). Muscles were activated in pairs by stimulating contralateral tibial nerves in state 1 and state 2 respectively. The broken grey lines in the insert represent the measurement times after stretch initiation including mechanical only component (10 milliseconds ms), dynamic time point (end of the ramp phase, 50ms), and static time point150 (end of the hold phase, 150ms).

A standard precollicular decerebration was completed in all cats (Silverman et al. 2005). This involved a vertical transection starting at the anterior margin of the superior colliculus. All brain matter rostral to the transection was removed. Gelfoam and cotton were placed in the cranium to control bleeding. After the decerebration, isoflurane anesthesia was titrated down over 15–30 minutes and withdrawn. In some cases, ventilation was required following the decerebration.

Data Acquisition

Intermuscular spinal reflex pathways were examined using a ramp-hold-release muscle stretch protocol. The details of the hardware and software have been described previously (Ross and Nichols, 2009). In brief, the protocol involved 2 mm muscle stretches with a 50 ms ramp, 100 ms hold and 50 ms release. The muscle stretches for a given trial were applied in a 2-state alternating pattern with a stretch repetition frequency of 0.7 Hz for a duration of 30 to 40 seconds (i.e. ~10–15 repetitions per state). In state 1, a single muscle referred to as the recipient was stretched. In state 2, the recipient muscle was stretched simultaneously with another muscle referred to as the donor muscle. Muscle stretch repetitions were applied with the recipient and donor muscles background forces set at a constant ~1–3 N, or over a range of background forces by eliciting a crossed extension reflex (XER). The crossed extension reflex was elicited by stimulating the tibial nerve just proximal to the medial malleolus with a nerve cuff or hook electrode (0.1 ms pulses, 40 Hz, 20–40 s duration). Typically, forces in the contralateral extensor muscles increase rapidly, plateau and slowly decay toward the original 1–3 N background force over a 20–40 seconds time period.

Data Analysis and Statistics

All data were analyzed using custom programs written in Matlab (Mathworks, Natick, MA). To account for varying background forces and identify the relative force change in response to applied stretches, a baseline force trajectory for each stretch repetition in a trial was subtracted from the raw force profiles prior to analyses. The baseline force trajectories used to remove the background force offset were determined using linear interpolation from stretch onset to a point 900 ms after stretch onset (Ross and Nichols, 2009, Lyle and Nichols, 2018). Stretch repetitions that had a sudden background force change precluding an accurate baseline force vector determination were eliminated from analysis, as were spontaneous force responses unrelated to muscle stretch or tibial nerve stimulation during a trial.

The dependent variable was the background force subtracted recipient muscle forces in response to the ramp-hold-release stretches. Force responses were measured 10 ms after initiation of the ramp stretch to check for mechanical artifacts, since length and force feedback require 18 – 20 ms to influence muscle force output. Mechanical artifacts are usually manifested by unloading responses resembling inhibition, so apparent inhibition at a latency of 10 ms would indicate unloading of the recipient muscle by the donor. Force responses were also measured at the end of the ramp phase (dynamic response) and hold phase (static response) to assess the development of inhibition as a function of time following the ramp. Then, the average muscle force response for a period 110–150 ms after stretch onset (i.e. last 40 ms of hold phase) was used to test all hypotheses in this study. The force values during this period have previously been shown to illustrate the intermuscular sensory feedback effects from a donor muscle onto recipient muscle (Lyle and Nichols, 2018). Intermuscular effects attributed to tendon organs were identified as the donor muscle reducing the recipient muscle stretch evoked force responses. For trials with stretch repetitions recorded with stable background forces, Wilcoxon rank-sum tests were completed on individual trials used to determine whether the recipient muscle forces recorded when stretched alone (state 1) were different from that when stretched at the same time with the donor muscle (state 2). The relative percent differences between recipient muscle forces obtained in states 1 and 2 were calculated in an effort to ease across limb and cat comparisons. Wilcoxon signed rank tests were used to examine whether the relative inhibition was different from FHL onto G vs G onto FHL in both limbs and whether FHL onto G and G onto FHL was different between the ipsilateral and contralateral limbs.

Intermuscular reflex pathways were assessed over a range of background forces by stimulating the contralateral tibial nerve. This protocol resulted in a population of force responses for state 1 and state 2. The populations of force responses for each state were plotted separately as a function of background forces. Background force values used in the regression plots were the muscle force means for the 15 ms period prior to stretch onset for each stretch repetition. The force responses were then fit with least squares quadratic polynomial curves. A regression model was used to evaluate whether the state 1 and state 2 force responses could be fit with the same curve or were statistically different warranting separate polynomial curve fit lines (Kutner et al., 2005; Ross and Nichols, 2009; Lyle et al., 2016). This was done by performing an F test using full and reduced regression models. The dependent variable was the stretch evoked force responses. The full model predictor variables included a grouping variable (state 1 and state 2), background force, background force squared, background force × grouping variable crossed term, and background force squared × grouping variable crossed term. The reduced model lacked the grouping variable terms. The F test evaluated the null hypothesis that the population of forces from state 1 and state 2 are the same and should be fit with a single polynomial fit line. In contrast, a p value ≤ 0.01 from the F test indicates a significant separation of the force populations, suggesting intermuscular spinal pathways from the donor muscle influenced recipient motor output. In addition to testing for significant separation with the F test, the relative magnitude of any difference between the polynomial curve fit vectors from states 1 and 2 were expressed as a percent difference [(state 2 curve fit vector – state 1 curve fit vector) / state 1 curve fit vector *100]. The polynomial curve fit difference was only computed across background force ranges spanned by both states. The difference between state 1 and state 2 was expressed as a relative percent to facilitate comparisons across muscle pairs and cats. The curve fit difference curves for each cat are being used in this study to characterize the strength of force feedback. In a previous paper, we completed post-hoc testing using curve fit difference data (Lyle and Nichols, 2019). Post-hoc testing was not completed in this study, in part, due to lack of overlapping background force ranges across cats. Mechanical artifacts were detected as described previously (Lyle and Nichols, 2018). Differences in force response between states 1 and 2 measured 10 ms after initiation of the ramp were deemed due to mechanical artifacts.

At the end of each terminal experiment, cats were euthanized either with an overdose of Nembutal followed by a pneumothorax (controls) or given isoflurane until unresponsive to stimuli followed by transcardial perfusion (cats with spinal hemisection). Prior to euthanasia in the cats with LSH, heparin (1cc;1000U/i.v.) was administered followed by 1cc of 1% sodium nitrite IV after 20 minutes. Transcardial perfusion was completed with 0.9% saline, followed by 4% paraformaldehyde in 0.1M phosphate buffer (PB; pH 7.4). The lesion segments were cryoprotected (30% sucrose in PB) and cut on a cryostat at 25 μms. Series of 1 out of every 10 sections were processed using Nissl (cresyl violet with acetate, Sigma-Aldrich, St. Louis, MO) and/or myelin stains (Eriochrome Cyanine R; Fluka, New York, NY). These stained sections were used to evaluate the extent of tissue sparing and damage. Tissue was screened to determine the section showing the greatest areal extent of damage. In combination with this section, multiple stained sections rostral and caudal were viewed and used to determine the areas of tissue disruption in each animal. This information was then combined and represented on a template outline of a spinal cord cross-section for each animal (Figure 2).

Figure 2.

Extent of the lesions in animals with sub-chronic and chronic LSH. Cross-sectional representations of lateral spinal hemisections. Areas of tissue damage and sparing for each animal were summarized onto a low thoracic spinal cord line template (a). Summaries were based upon assessment of multiple Nissl and myelin stained sections at and within several hundred microns of the lesion epicenter in each animal. An example of a stained tissue cross section at the lesion epicenter of one animal is shown (b) along with its summary lesion composite (d). There is some variation at the midline across lesions. This variation includes partial sparing of the ipsilesional medial ventral funiculus in two animals (f, g) and contralateral, partial gray matter damage primarily in the intermediate gray matter of another (h). Despite these differences, the lesions are relatively uniform, disrupting half of the spinal cord. Time points are 2 weeks (c), 4 weeks (d-f), 8 weeks (g) and 20 weeks (h) post-injury. Micrometer bar = 500μms

RESULTS

Spinal cord lesion consistency

All lesions were verified to be at the T9 or T10 spinal level based upon gross anatomical root counts and distinctive dorsal root ganglia morphologies. Histological sections showed that injuries generally disrupted the ipsilateral white and gray matter with some minor variations in the six animals with sub-chronic or chronic thoracic hemisections (Figure 2). With respect to sparing of ipsilesional, long, descending supraspinal tracts, partial medial sparing of the ventral, white matter area was seen in one of the animals 4 weeks (Figure 2f), and the single animal 8 weeks, following spinal cord injury (Figure 2g). Overall, hemisection lesion variation was minimal across the animals. Lesions were not assessed in the two animals with acute injuries made during the terminal studies as those animals were not transcardially perfused.

Intermuscular inhibitory Force feedback between FHL and G

Active Trials

A representative active trial for control animals is shown in Figure 3. The force and length traces for G and FHL with G as the recipient are shown in Figure 3 (f, g), and the force and length traces for FHL as the recipient in Figure 3 (c, d). The force and length traces for G and FHL as the donor muscle are shown in Figure 3 (a, b, h, i) respectively. Note that the force responses of the recipient muscle are reduced in state 2 for both directions over the range of forces, indicating bidirectional inhibition. Panels 3e and 3j show individual pairs of responses for states 1 (dark line) and 2 (light line) respectively, obtained at similar background forces. The inhibition can generally be detected early during the ramp phase and is well developed at the dynamic time point (Bonasera and Nichols, 1994, 1996; Wilmink and Nichols, 2003), suggesting a contribution from Ib afferents (Nichols, 1999). In agreement with previous studies, the inhibition was found to decrease with decreasing background force.

Figure 3.

Representative control data. Ramp and hold stretches were delivered to the recipient muscle (force traces: c, f; length traces: d, g) on every stretch repetition (state 1: solid black squares above recipient muscle force trace), and also to the donor muscle (force traces: a, h; length traces: b, i) on every other stretch repetition (state 2: grey circles above recipient muscle force trace) as background force slowly declined after a crossed-extension reflex. The left column shows the influence of donor G (a) onto recipient FHL (c). The right column shows the influence of donor FHL (h) onto recipient G (f). The response of the recipient FHL (c) and G (f) force traces is reduced for state 2 stretches. The bottom two traces show single stretch responses of FHL (e) and G (j) from state 1 (dark line) and state 2 (light line) superimposed, illustrating the time course of the inhibition. Note that the inhibition was bidirectional in this case. *FHL, flexor hallucis longus; G, gastrocnemius.

Figure 4 illustrates the analysis of data shown in Figure 3, with force responses from FHL onto G shown in the top panels (a, b, c) and G onto FHL in the bottom panels (d, e, f). Data points for state 1 were fitted with quadratic polynomials (dark line) and plotted along with 95% confidence limits. Quadratic fits for state 2 data are shown with light lines. The left column (a, d) portrays the force responses recorded 10 ms after the onset of stretch (mechanical response). Small or insignificant differences were noted in both directions for G and FHL respectively indicating that there were no significant mechanical artifacts. The second column (b, e) of responses were recorded at the end of the ramp (dynamic response), and show that the inhibition increased with increasing background force. The inhibition was fully developed at the end of the hold phase (static response), as indicated in the right column, FHL onto G (c, p<0.01) and G onto FHL (f, p<0.01), respectively (see Table 1 for additional statistical details).

Figure 4.

Analysis of representative control data. Force responses were obtained during decaying crossed-extension reflexes. Each response was measured at three time points during the stretch and these measurements were each plotted against the background forces evoked by the crossed-extension reflex. The measurement times after stretch initiation were 10 milliseconds (ms) (mechanical only component: a, d), 50 ms (end of the ramp phase/dynamic time point: b, c), and 150 ms (end of the hold phase/ static time point: c, f). Top panels (a, b, c) are for trials with G as a recipient, and bottom panels (d, e, f) with FHL as recipient respectively. Data points for state 1 were fitted with quadratic polynomials (dark line) and plotted along with 95% confidence limits. Quadratic fits for state 2 data are shown with light lines. Populations of data were distinctly separated for both dynamic and static phases G and FHL, respectively. The absence of significant differences for the 10ms time point for G and FHL respectively, indicates that there were no significant mechanical artifacts. Black squares represent force responses of recipient muscle from stretches occurring in state one and grey circles in state two, respectively. Note that the inhibition is better developed for the static time point. *FHL, flexor hallucis longus; G, gastrocnemius.

Table 1.

Table 1 shows statistical details for intermuscular inhibition between FHL and G shown in Figure 4 (cat with chronic LSH) and Figure 6 (control cat).

Cats	Muscle interactions	F value	P value
Control	G onto FHL	F3,40 = 25.1	< 0.01
	FHL onto G	F3,41 = 339.9	< 0.001
Chronic LSH	G onto FHL	F3,35 = 84.8	< 0.001
	FHL onto G	F3,40 = 22	< 0.001

Following LSH, the inhibition was amplified and biased from FHL onto G, and reduced from G onto FHL as illustrated in Figures 5 and 6 irrespective of the time post SCI. Figure 5 shows raw data and averaged responses for force feedback between FHL and G in an animal with chronic LSH 4 weeks post injury (cat 3). The force and length traces for G and FHL with G as the recipient are shown in Figure 5 (f, g), and the force and length traces for FHL as the recipient in Figure 5 (c, d). The force and length traces for G and FHL as the donor muscle are shown in Figure 5 (a, b, h, i) respectively. Note the inhibition visible in the G force record (a) when FHL was stretched alone (c). The inhibition is evident for both dynamic and static responses (e), but was particularly pronounced at the static time point (j). The rapidly developing inhibition during the hold phase of the ramp can be appreciated from the pair of responses shown in Figure 5j. The polynomial force plots corresponding to these data are shown in Figure 6. As was the case for the data shown in Figure 4, there was no evidence for significant mechanical interactions during the mechanical time point (a, d). Inhibition from FHL onto G for the dynamic time point (b, e) was indicated by the separation of the two groups of points. The inhibition for the static time point was large for FHL onto G (c, p<0.01). On the other hand modest inhibition from G onto FHL was observed at the static time point (f, p<0.01). Further statistical detail is given in Table 1.

Figure 5.

Representative data for an animal with LSH, injured 4 weeks before the terminal experiment (chronic LSH). Data presentation similar to that in Figure 3. The left column shows the influence of donor G (a) onto recipient FHL (c). The right column shows the influence of donor FHL (h) onto recipient G (f). The bottom two traces show single stretch responses of FHL (e) and G (j) from state 1 (dark line) and state 2 (light line) superimposed, illustrating the time course of the inhibition. Note the strong inhibition from FHL onto G (f) and a weak inhibition of G onto FHL (c). (j) Reflex latency for the FHL/G interaction was calculated at 28 ± 4 ms comparable to control animals shown in figure 3. There was no evidence of autogenic inhibition in state 1 and the force of recipient muscle did not drop. *FHL, flexor hallucis longus; G, gastrocnemius.

Figure 6.

Analysis of data from animal with 4 week spinal hemisection (Chronic LSH). Data presentation is similar to that in Figure 4. The inhibition for the static time point (c) was large for FHL onto G as seen by the significant vertical separation of the state 1 (dark) and state 2 (light) fitted polynomial curves. In contrast, modest inhibition was observed from G onto FHL (f) at the static time point as seen by close state 1 (dark) and state 2 (light) fitted polynomial curves. Note the directional bias from FHL onto G. This pattern was observed in all animals with LSH (1/1 sub-chronic and 5/5 chronic). *FHL, flexor hallucis longus; G, gastrocnemius.

In order to compare results across animals and between ipsilateral and contralateral limbs, force responses for the last 40 ms of the hold phase were averaged and force plots constructed using these averages. The force plots corresponding to states 1 and 2 were then subtracted and expressed as relative percent differences to yield curves representing the differences across background forces (Figure 7). These difference curves were then plotted together across experiments (represented by different colors). Data evaluating the influence of FHL onto G are shown as solid lines, and data for G onto FHL are shown as dotted lines. Directionally biased inhibitions are represented by vertically separated difference curves (solid and doted lines separation) for each animal. Data from the same experiment are indicated by lines with the same color in ipsilateral (LSH side limb) and contralateral limbs. The data from animals with chronic and sub-chronic LSH are illustrated in Figures 7a (ipsilateral limb) and 7b (contralateral limb). Cat 6 does not have contralateral limb data due to lack of an effective crossed extension reflex on this side during the experiment. The extent of the inhibition clearly varied substantially from animal to animal. The inhibitions from G onto FHL, however, were restricted to a small, narrow range both on the ipsilateral limb (a, dotted lines all p <0.01) and contralateral limb (b, dotted lines all p <0.01). The inhibition from FHL onto G (a and b, solid lines all p <0.01) was larger in all cases but the contralateral limb in cat 2 which had similar inhibition bidirectionally. The directional bias in inhibition from FHL toward G was observed without clear influence of time since spinal injury. The inhibition was generally stronger on the ipsilateral limb (a, solid lines all p <0.01) in comparison to the inhibition on the contralateral limb (b, solid lines all p <0.01). Further statistical details are presented in Table 2.

Table 2.

Table 2 shows statistical details for intermuscular inhibition between FHL and G data shown in Figure 7 (cats with chronic/sub-chronic LSH and control cats) and Figure 8 (acute LSH).

Cats	G onto FHL				FHL onto G
	F value	P value	F value	P value	F value	P value	F value	P value
Control	Lt Limb		Rt Limb		Lt Limb		Rt Limb
Cat 1	F3,29 = 203.8	< 0.001	F3,34 = 53.7	< 0.01	F3,31 = 33.6	<0.01	F3,27 = 26.7	< 0.01
Cat 2	F3,37 = 38.3	< 0.01	F3,30 = 21.8	< 0.01	F3,36 = 67.3	<0.001	F3,28 = 76	< 0.01
Cat 3	F3,42 = 38.3	< 0.001			F3,43 = 14.5	< 0.01
Cat 4	F3,37 = 12.7	< 0.01	F3,41 = 41.2	< 0.01	F3,39 = 352.4	<0.001	F3,28 = 76	< 0.001
Cat 5	F3,40 = 61.1	< 0.001	F3,42 = 17.1	< 0.01	F3,41 = 12.9	< 0.01	F3,43 = 51.4	< 0.001
Acute LSH	Ipsilateral Limb		Contralateral Limb		Ipsilateral Limb		Contralateral Limb
Cat 1	F3,27 = 4.7	< 0.01	F3,28 = 6.2	< 0.01	F3,28 = 199.8	<0.001	F3,47 = 100.4	< 0.001
Cat 2	F3,21 = 1.7	< 0.01	F3,22 = 1.1	< 0.01	F3,24 = 15.7	< 0.001	F3,25 = 34	< 0.001
Chronic LSH	Ipsilateral Limb		Contralateral Limb		Ipsilateral Limb		Contralateral Limb
Cat 1 (2W)	F3,31 = 15.5	< 0.01	F3,14 = 215.3	< 0.01	F3,18 = 917	<0.001	F3,16 = 558.8	< 0.001
Cat 2 (4W)	F3,14 = 29.5	< 0.01	F3,13 = 47.5	< 0.01	F3,14 = 204.7	<0.001	F3,15 = 35.4	< 0.001
Cat 3 (4W)	F3,47 = 61.6	< 0.01	F3,40 = 25.4	< 0.01	F3,42 = 437.4	<0.001	F3,52 = 285.5	< 0.001
Cat 4 (4W)	F3,38 = 31.5	< 0.01	F3,46 = 68.4	< 0.01	F3,38 = 106.8	<0.001	F3,38 = 58.6	< 0.001
Cat 5 (8W)	F3,36 = 4.3	< 0.01	F3,24 = 5.1	< 0.01	F3,41 = 139.6	<0.001	F3,30 = 259.8	< 0.001
Cat 6 (20W)	F3,23 = 3.6	< 0.01			F3,23 = 65.8	<0.001		< 0.001

For comparison, difference curves derived from control data are shown in Figures 7c (left limb, all lines p<0.01) and 7d (right limb, all lines p <0.01). The relative inhibitions were either balanced between FHL and G, or the relative inhibition was biased with stronger inhibition from FHL onto G or stronger inhibition from G onto FHL, as shown previously (Bonasera and Nichols, 1994; Lyle and Nichols, 2018). Balanced inhibitions are represented by approximate coincidence of difference curves, and directionally biased inhibitions are represented by vertical separation between the solid and dotted difference curves. Similar results were found for both the left and right limbs in each control animal as evident by statistical details described in Table 2. Although the ranges of inhibition were similar for control animals and those with LSH, a clear bias of inhibition from FHL onto G was consistently observed only in animals with LSH.

The data from the animals with chronic lesions indicate little change in the pattern of inhibition over survival times. In order to further test whether the characteristic pattern after partial spinal cord injury was a release phenomenon rather than a result of plasticity, we performed two experiments in which the spinal lesion was made during terminal experiments (acute LSH). The results from these acute LSH experiments are shown in Figure 8 and illustrate essentially the same pattern as observed in the chronic studies. A clear bias of inhibition from FHL onto G was observed across animals with acute LSH (all solid lines p<0.01) when compared to the inhibition from G onto FHL (all dotted lines p<0.01) in both the ipsilateral (a) and contralateral (b) limbs (Table 2 for detailed statistics)

Figure 8.

Pattern of force-dependent inhibition following an acute LSH. Data from two animals are shown in two different colors. The format is the same as that in Figure 7. In both cats, inhibition was greater from FHL onto G (solid lines) compared to G onto FHL (dotted lines) consistent with the pattern observed with chronic spinal cord injury. *FHL, flexor hallucis longus; G, gastrocnemius.

Quiescent Trials.

For trials in which the crossed extension reflex (XER) was not evoked in animals with chronic LSH, forces remained low and relatively constant. In these cases, the force responses from the state 1 and 2 stretch repetitions (e.g. 10–15 per state) were averaged separately for each animal in each trial and expressed as a % change from state 1 to assess the strength of inhibition. Shown in Figure 9 are boxplots with dotplot overlays summarizing the relative inhibition from FHL onto G and G onto FHL in the ipsilateral and contralateral limbs of cats with chronic LSH. The inhibition from FHL onto G was statistically greater when compared to G onto FHL for both limbs (Wilcoxon signed-rank tests: ipsilateral: z=2.0, p=0.04 and contralateral: z=2.0, p=0.04). There was no difference when comparing between ipsilateral and contralateral limbs for either FHL onto G or G onto FHL (Wilcoxon signed-rank tests: p > 0.05 for both). These data are in general agreement with the active trials reported above.

Intermuscular inhibitory Force feedback between FHL and SOL, PLANT

The purpose of studying force feedback interactions between SOL, PLANT and FHL was to further determine the distribution of the intermuscular FFB interactions among ankle extensor muscles in the hindlimb of the cat after LSH. We also wanted to examine whether the reorganization of intermuscular interactions in LSH was limited to G and FHL. Force feedback pathways between these muscles are inhibitory under conditions of quiet stance (Bonasera and Nichols 1994; Nichols, 1999; Wilmink and Nichols, 2003). The ankle extensors SOL and PLANT also received strong, essentially one-way inhibition from FHL in animals with LSH, in contrast to the variable directional biases observed in control animals (Bonasera and Nichols, 1994; Nichols, 1999; Wilmink and Nichols, 2003; Lyle and Nichols, 2018). Representative difference curves, for both legs in an animal with chronic LSH, are shown in Figure 10. Note that the inhibitions onto FHL from both SOL (dotted dark lines) and PLANT (dotted light lines) were negligible (p > 0.01), while the inhibitions from FHL onto the two other extensors (SOL= solid dark line and PLANT= solid light line) are large, particularly for the leg ipsilateral to the lesion (ipsilateral and contralateral solid lines p<0.01). Detailed statistics for figure 10 are given in Table 3. Similar results were obtained in 3 of 3 cats tested (cats 3, 4 & 6) for FHL onto SOL at p ≤ 0.01 and 5 of 5 cats (cats 1, 2, 3, 4 & 6) for FHL onto PLANT at p ≤ 0.01 bilaterally (data not shown). We did not collect data from the SOL muscle in three experiments (cats 1, 2 & 5) and for PLANT in one experiment (cat 5). Similar results were obtained in two cats with acute LSH in both ipsilateral and contralateral limb (data not shown).

Figure 10.

Representative results for FHL, SOL and PLANT. Difference curves (see Figure 7) were plotted for FHL as recipient (dashed lines), and SOL or PLANT as recipient (solid lines). The inhibition onto FHL was negligible but large onto SOL or PLANT. In addition, the inhibition onto SOL or PLANT was greater for the ipsilateral leg (a) in comparison to the contralateral leg (b). Similar results were obtained in 3 of 3 cases for FHL and SOL, and 5 of 5 other cases for FHL and PLANT (data not shown) in animals with chronic LSH.

Table 3.

Table 3 shows statistical analysis for data represented in Figure 10 showing intermuscular interactions between FHL, Sol and PLANT in a cat with chronic LSH (cat-4, terminal experiment performed 4 weeks post LSH)

Chronic LSH	Muscle Interaction	Ipsilateral Limb		Contralateral Limb
		F value	P value	F value	P value
Cat 4 (4W)	SOL onto FHL	F3,44 = 4.4	< 0.01	F3,44 = 1.8	0.01
	FHL onto SOL	F3,49 = 277.2	< 0.001	F3,38 = 769,6	< 0.001
	PLANT onto FHL	F3,44 = 0.15	0.9	F3,45 = 1.1	0.9
	FHL onto PLANT	F3,43 = 1589.7	< 0.001	F3,43 = 326.2	< 0.001

Clasp knife inhibition

No evidence of clasp knife inhibition was observed among G, SOL, PLANT and FHL in animals (6/6) following chronic LSH in contrast to its presence documented earlier in complete spinal transection (Nichols and Cope 2001). The heterogenic inhibition in state 1 and 2 was observed even at very low background forces of the recipient muscle (Figure 5). Figure 5 (e, j) shows a representative example of the absence of any autogenic inhibition between G and FHL following LSH, depicted by the absence of force drop in state 1 (dark line) during the muscle stretch. The hallmark of clasp knife inhibition after complete spinal cord transection has been described as a profound autogenic and heterogenic inhibition that occurs with a latency of more than 80 ms (Nichols and Cope 2001). The latency of reflex responses (e.g. Figure 5, <30 ms) observed was comparable to decerebrate cats with intact spinal cords (Bonasera and Nichols 1994). In addition, clasp knife inhibition was not observed in the acute LSH experiments.

Discussion

Main Findings

We investigated the strength and distribution of inhibitory force feedback between the long toe flexor FHL and the ankle extensors G, SOL and PLANT in animals with LSH (chronic, sub-chronic and acute) and compared these results with those from an untreated control group using the same methods. These studies were conducted with the animals in the decerebrate state. In control animals, we observed the expected variability in the magnitude of inhibition and the variability in directional bias of the inhibition between FHL and G across preparations. Using electrophysiological methods, Eccles and coworkers (1959) indicated that SCI causes an amplification of inhibitory feedback over that observed in the control decerebrate state. In contrast to these earlier observations and more in line with the more recent demonstrations of complex changes in spinal pathways following injury (Rossignol and Frigon, 2011), we observed a reorganization of the weightings of inhibition rather than a generalized increased in strength of the inhibition. Inhibition was increased in some directions and diminished in others. We found a pronounced directional bias from FHL onto G, SOL and PLANT in all cases for the limb ipsilateral to the LSH, and the same bias in the contralateral limb as well as an amplification of the inhibition in the preferred direction. The observed changes in the pattern of force feedback was independent of survival time following spinal cord injury, suggesting that the reorganization of intermuscular inhibition resulted mainly from a release from descending control rather than a developing adaptive process.

Comparison with previous research on the force feedback network

It was shown in the earlier electrophysiological studies that a long toe flexor is a particularly strong source of inhibition to other ankle extensors in the decerebrate state (Eccles et al., 1957b) and that this inhibition is greatly increased following spinal transection (Eccles and Lundberg, 1959). Although this muscle was identified as flexor digitorum longus (FDL) by the Eccles group, results from our laboratory using muscle stretch (Bonasera and Nichols, 1994; Nichols, 1994) indicated that FHL rather than FDL is the main source of inhibition from the long toe flexors. Since electrical stimulation was used in the earlier study, it is likely that the nerves from both muscles were stimulated due to current spread. The results reported here indicate that indeed, inhibition from FHL is amplified following LSH, in agreement with the earlier studies. However, the inhibition from the other ankle extensors onto FHL was quite variable in control decerebrate animals (Lyle and Nichols, 2018), and in some cases, the inhibition from FHL was substantial. We therefore interpret our results as evidence that, rather than a simple amplification of inhibition, the spinal cord injury replaces the variable patterns of inhibition with a single, directionally biased pattern, in which the inhibition from FHL onto ankle extensors is amplified over that observed in control preparations.

A more uniform pattern of inhibition also was observed during stepping in premammillary preparations (Ross and Nichols, 2009), a more defined behavioral state than may be the case for the precollicular decerebrate animal. In the premammillary preparation during stepping, the pattern of inhibition corresponded largely to a proximal to distal gradient, focusing inhibition on the distal musculature. This pattern contrasts markedly with the pattern observed here after spinal cord injury, and suggests that the three patterns observed in the control decerebrate animals with precollicular transections (Lyle and Nichols, 2018) represent different states of the spinal motor apparatus. A direct comparison of the patterns of inhibition with and without spinal hemisection in the stepping premammillary preparation would be desirable and is planned for the near future. Although the data from the stepping preparations show a consistent pattern, the yield from such experiments is much lower than with the precollicular decerebration, precluding using this approach exclusively. Furthermore, the use of the precollicular transection is an important starting point to establish a robust body of data on which to base future studies. Three distinct patterns were observed that may provide a foundation on which to classify the patterns relevant to distinct behavioral states. Under development is a technique that will allow mapping of intermuscular inhibition and excitation in the intact, behaving animal (Pratt, 1995; Lyle and Nichols, 2019). This less invasive method, although less quantitative than the terminal experiments described here, will allow the investigation of the force feedback system during a variety of behaviors and over the time course following neurological injury.

Potential receptor contributions

The results of previous studies of intermuscular inhibition indicated that the inhibition was mediated by afferents from Golgi tendon organs (Bonasera and Nichols, 1994; Wilmink and Nichols, 2003; Nichols, 1999). This conclusion was based on the force dependence of the inhibition and sensitivity to strychnine, indicating glycinergic transmission. In the studies reported here, the inhibition in the stronger directions was force dependent, and also larger at low forces than typically observed in control animals. The larger inhibition at lower forces suggests either increased transmission through tendon organ pathways, or the addition of inhibition through pathways arising from other receptors. One candidate is the group II spindle afferent. Pathways from these receptors can be inhibitory and in addition, interneurons in the deep dorsal horn receive convergent input from both Ib and group II afferents (Jankowska and Edgley, 2010). Given the release of pathways that can occur with spinal cord injury (Eccles and Lundberg, 1959; Frigon et al, 2011), it is possible that the increased inhibition observed here was due in part to the increased participation of group II pathways. Studies to distinguish the relative contributions of group Ib and II pathways are currently underway.

Potential pathways regulating the force feedback network

In agreement with earlier studies (Cleland and Rymer, 1990), recent preliminary data (Niazi et al., 2012, 2014; Niazi, 2015) indicate that dorsal hemisection (disruption of the dorsal half of the spinal cord) leads to the expression of clasp-knife inhibition but leaves the organization and strength of force-dependent inhibition unaltered from that observed in animals with intact spinal cords. We provisionally conclude from these data that the organization of force feedback is regulated by pathways in the ventral funiculi. Other data from our laboratory has shown that signals arising from the vestibular system and neck afferents, that represent body orientation (Magnus and Deklejn, 1912; Roberts, 1967; Wilson, 2013), influence central pattern generating circuits (Gottschall and Nichols, 2009) and also the strength of intermuscular inhibition in a task-dependent manner (Nichols et al., 2014). The body orientation signal is mediated at least in part by the vestibulospinal tract. These results taken together indicate that the organization of force feedback is likely to be regulated by signals descending in the vestibulospinal and possibly reticulospinal tracts, and therefore affected by ventral section of the spinal cord. These two pathways, however, are thought to project ipsilaterally (Carpenter and Sutin, 1981) so the bilateral effect we observed was unexpected. This observation suggests that the bilateral effect was mediated by as yet undescribed commissural pathways in the spinal segment. It has recently been shown in the rat that the reticulospinal pathway contacts local commissural cells in the cervical spinal cord and also long descending propriospinal neurons that may project ipsilaterally or contralaterally (Mitchell et al., 2016), suggesting the reticulospinal pathway as an additional candidate controlling the force feedback network.

As stated above, the loss of descending control leads to a change in inhibitory strength between muscles. It may also be asked whether there is a change in which muscle combinations are linked by the inhibition following spinal cord injury. Although the sample of muscles was too limited in this study to provide a comprehensive answer to this question, the inhibition became more biased toward the triceps surae and PLANT following injury but FHL still received some inhibition, showing that there was no change in which muscles received inhibition. Subsequent studies will address this question by investigating a larger number of muscles in the hindlimb. Length feedback pathways, which are more focused in the uninjured spinal cord, show widespread effects following complete spinal transection (Heckman, Hyngstrom and Johnson, 2008; Johnson et al., 2013). Inhibitory force feedback is already widespread in the control case and remains so after spinal hemisection, but the way the inhibition is distributed changes.

Functional implications

FHL flexes the toes and metatarsophalangeal joint and plantarflexes the ankle, while the triceps surae muscles flex the knee to a small extent and extend the ankle. PLANT flexes the knee to a small extent, plantarflexes the ankle, and flexes the metatarsophalangeal joint (Goslow et al., 1972). During spontaneous stepping movements in premammillary decerebrate cats (Ross and Nichols, 2009), intermuscular, force-dependent inhibition was found to be distributed mainly from proximal to distal muscles, including from the triceps surae muscles onto FHL and quadriceps to the triceps surae muscles. A functional interpretation of this organization is that the inhibition would increase the compliance of distal joints (ankle and metatarsophalangeal) that interact most directly with the environment, providing the appropriate mechanical interface (Nichols, 2018; Simon et al., 2014) for locomotion. In non-locomoting, decerebrate animals, a variety of patterns of inhibitory feedback were observed (Lyle and Nichols, 2018), as noted above, including a pattern similar to that observed during spontaneous stepping (Ross and Nichols, 2009). The consistent observation in the present studies of increased inhibition from FHL onto ankle extensors and reduced in the opposite direction suggests that the ankle might be more, and the metatarsophalangeal joint less, compliant than normal. This pattern would have the effect of reducing the mechanical coupling between ankle and knee, and therefore altering the normal proportional coordination between these joints (Goslow et al., 1973; Abelew et al., 2000). Studies addressing possible correlations between the observed changes in the organization of proprioceptive pathways and behavior following spinal cord injury are underway.

SIGNIFICANCE STATEMENT.

Inhibitory force feedback from Golgi tendon organs is exchanged between lower limb muscles and thus can influence motor output. Evidence indicates the strength and distribution of inhibitory force feedback between muscles is modulated by supraspinal structures according to task. This study demonstrates, in contrast to control animals, a consistent pattern of Golgi tendon organ feedback after spinal hemisection in the decerebrate animal that may correspond with impairments observed during walking. These data implicate altered feedback from Golgi tendon organs after spinal hemisection as a potential explanation for motor impairments warranting further examination.