CNS Axon Regeneration in the Long Primary Afferent System in E15/E16 |Hypoxic-Conditioned Fetal Rats: A Thrust-Driven Concept

1. Introduction

An incidence increase of severe spinal cord injury among older people is a worldwide trend. Older people with SCI risk more complications. Data from more than a thousand clinical trials shows that recovery characteristics have remained unchanged over the last twenty years [1,2,3]. The pattern of neurological and functional recovery of older people might have counterbalanced positive effects from approved care in acute and rehabilitation practices. Given the wealth of knowledge of the central nervous system existing today, medical science seems hesitant to pay off its merits in SCI. Major domains relevant to CNS repair and regeneration, like Biochemistry and Neurobiology, have enriched scientists' armamentarium to tackle life's intricate mechanisms inherited from phylogenetically lower species capable of cell and tissue regeneration [4,5,6]. The experienced incapability of the mature CNS deviated science from the lesion site after "a century of sterile endeavor"[7]. However, cell-based transplantation experiments in higher vertebrates pulled back with promising results [8,9]. The first Phase I safety studies of human autologous Schwann cell transplantation in patients with SCI and human fetal neural stem cell transplants for chronic SCI were launched in 2012 and 2013 [8,10]. Since then, research papers on non-human or human-induced pluripotent stem cells have peaked. So far, various cell suspensions are delivered mostly by intrathecal injections and have reached Phase I/II clinical trials for SCI worldwide [11]. Small patient numbers and short follow-up periods hamper assessing the immediate effects of very early interventions, i.e., ≤ 2 weeks post-injury [1]. Preclinical studies demonstrate this incapacity of axon outgrowth that extends beyond the CNS lesion for a 10 mm length max and after days or weeks.

We might classify these features as sprouting like central axons, creating collaterals (colls) at their final stage of axon development [5,12]. The incapacity of CNS regeneration is ascribed to the mammalian dorsal root ganglia neuron’s lack of long-distance axon elongation across the white matter. Since the turn of the century, this notion has been ranked a law of nature in neuroscience [7,13]. The inhibitory environment of the lesioned adult spinal cord is thought to prevent the expression of the innate ability. The neuron with its pseudounipolar axon can regenerate its cut peripheral branch long distance. The acute primary and lasting secondary effects in experimental spinal cord transections ruin the tissue's cytoarchitecture, demonstrating central axon regeneration failure. For decades, research into extrinsic factors like myelin and proteoglycans challenged glial scar formation [14]. Meanwhile, the human-induced progenitor stem cell-based transplantation paradigm came into practice [15]. Before that, the absence of glial scarring in the injured immature spinal cord had been incidentally investigated [16,17]. Research into immature CNS tissue for conditions conducive to CNS regeneration rendered conflicting results and hindered an expected breakthrough. Our conference report indicated an intrinsic factor precluding fetal spinal cord regeneration. In a small S-series of E17/E18.p42 rats, the glucose metabolism at the gracile nuclei was comparable to control [18]. These experiments revealed that the fetal dorsal myelotomies failed for expected signs of regeneration and were devoid of scar tissue. This initially observed baffling perspective prompted the endeavor to acquire more upstream targets. Facing financial constraints at the time ultimately postponed this contribution to neuroscience.

Standing at a crossroads, do we consider finding the maze’s way out while keeping the same track? On the one hand, the possibly negative impact of older people in the clinical trials could counterbalance a noticeable effect on recovery. On the other hand, questioning whether applied translational science might still account for improvements in neurological and functional recovery from current clinical trials is valid. Although many preclinical studies have claimed regeneration, this issue moves in circles regarding sprouting. We suggest an addition to the affirmative criteria for CNS regeneration, which was stated twenty years ago [19] (Discussion). Our recent paper documented the long primary afferent system development, focusing on Clarke’s and gracile nuclei [12]. Here, we present intriguing features of long primary afferent axons that regenerate and elongate presumably quickly towards their medullary targets after a single cut during fetal development.

2. Material and Methods

2.1. A Summary

The data presented in this paper was part of an excerpt of experimental work predating the European legislation on animal welfare and institutional ethics board approval. The animal facility and husbandry reflected the highest ethical standards applicable at that time. High fetal survival rates and many long-surviving rats justify this statement (Table 1). The pregnant rats were housed solitary in cages at 12:12 light cycle, having unlimited access to food and water. We witnessed derangements in the daily routine when rats had moved the unweaned litter from one corner to another during construction work in the facility. Increased anxiety, especially among primiparous rats, prompted us to postpone scheduled experiments. We summarize a short technical description here. For more details, we may refer the reader to our recent papers. The microsurgical dorsal myelotomy at random cervical and thoracic levels was aimed at disrupting the fetal rat's primary afferent system development. The dam's conception day was scheduled fifteen to seventeen days before at E0_, covering the long primary afferent system development in E15–E17 fetuses. The spontaneously breathing dam recovered within one or two minutes of quitting fluothane inhalation after the procedure, which took approximately half an hour. Applying the horse-radish peroxidase (HRP) tracer only to the left sciatic nerve's proximal stump was straightforward. The spinal cords had been sectioned sagittally for processing except for the horizontal sections of the medulla. Enzyme histochemistry involved tetramethylbenzidine (TMB), according to Mesulam [20]. Focused on the DC, we valued the tracing with HRP for its competent histology exhibiting the elongating axons in sagittal sections. This approach has enabled us to describe the assemblage of the intermediate and long subdivisions beyond the critical period (CP) in the previous paper [12]. Here, we present six experiments demonstrating the CP from a subset of cases that fulfilled all the sequential and hazardous processing steps. Given their places on the list ordered by estimating the M₀s on the assembly line, chances were challenged with perseverance, underlining that data acquisition was full of flaws and mishaps (Table 1). Uncovering the unknown phenotypes described recently yields a conclusive interpretation of the results aggregated for this paper. First, the next chapter recapitulates the long primary afferent system’s blueprint for the reader’s convenience [12].

2.2. Preamble to the Long Primary Afferent System’s Development

We have outlined the development cascade of the long primary afferent axons governed by the DRG neurons in the lumbar spinal column [12]. Using HRP tracing of the sciatic nerve, we hypothesize transition hubs (THs) on the assembly line of temporospatial axon development. Three or four consecutive stations are identifiable (Figure 1A). All the pioneering axons elongate in a highly dynamic TH.0 stage and reach the medulla in high tide waves. Axon tips at the medulla, taking jointly a very short time, transition into the TH.1 stage during a standing tide. Thereby, all upstream axons passing through this narrow time window down their imaginable assembly lines demonstrate the hypothetical fast elongation stop (Figure 1B, f-ES). At the second transition, they form less dynamic TH.2 stage collaterals (colls) penetrating the gracile nucleus. This phenomenon, the slow elongation switch (Figure 1B, s-ES), coincides with an elongation that parallels or synchronizes with spinal cord growth. Two instructive graphics displaying the actual development state before and after the myelotomy are added to grasp the actual states quickly (Figure 1C,D). Such graphics pertain to cases 3 to 8. Characteristics of the CP are highlighted in Figures 2A and 2B.

2.3. The Impact of Hypoxia during the Critical Period

The microsurgical dorsal myelotomy (Tx) is performed under compression of the placenta’s vasculature. The ensuing fetal tissue hypoxia induces reprogramming of the DRG neurons if done upstream at the proper M₀ (Glossary). This susceptibility is restricted temporally and upstream stage-dependent. The resulting perturbed phenotype mimics the imaginable upstream invisible TH.1 stage. In contrast to that naturally brief and undetectable axon feature, its mimicry remains visible under any circumstance and has a typical tracing feature. These neonatal axons exhibit the abrupt front stop (a-FS) when bundled. The single axons form the i-FSs. The bluntly tipped terminal club-like features always terminate in the white matter.

Our findings have revealed that a half-hour of oxygen shortage can reprogram DRG neurons. These alterations might link transcriptomic or translational perturbations documented in former research to HIF pathways, objectifying the pivotal impact of hypoxia [21,22,23,24]. The permanently visible i-FSs create stills of the naturally unstoppable primary afferent development cascade at various temporospatial spots on the assembly line. The stills have enabled us to distinguish between the high and low tide i-FSs and describe the dichotomous trajectory of the long primary afferent system. Those high and low tide axons build the long and intermediate subdivisions during and after the CP [12].

The CP encompasses the time involved in the long primary afferent axon elongation, in which a rostrocaudal gradient of axon development occurs. The smaller this window, the faster the growth, considering that halfway through the CP, 50% of the axons are still pioneers (Figure 2A). The smaller the time window, the more likely the Tx might render the exclusively monomorphic neonatal a-FS features. They are assigned to the pioneering axons' second half, originating in the L5 and, or L6 DRG neurons. In contrast, the Txs during the first half might render the multimorphic features with parent axons. Our data matches high-speed growth harmoniously with such considerations (Figure 2B).

During spring tide, the lumbar TH.0 axons pioneer toward the medulla in high tide waves [12]. Fortunately raised at an uncertain M₀, by chance precisely within the appropriate CP’s narrow time window, six bell-ringer cases showcase configurations exhibiting pertinent dynamic HRP features (Table 1). The feature mix reflects an -estimated- spot on the speculative development cascade and determines the order of the assembly line (Figure 2B, red-blue diagonal). An inferred reduction in available energy might explain the imminent decrease in axon elongation assigned to thrust (Discussion).

2.4. A Watershed Delineates the Critical Period

The cutting of pioneering TH.0 stage axons renders the distinct phenotype of neurons extending the clear i-FSs. These reprogrammed TH.1 stage mimicries exhibit permanent, thickened fiber terminations substituting the hypothetical transient and ephemeral TH.1 stage axon feature. Assumingly, the max visible i-FSs might entail 50% at spring tide. The appropriate upstream Txs render this monomorphic feature only discernable in neonates. The development block is demonstrated in the a-FSs and i-FSs. The highly dynamic nature of spring tide manifests in the axons that regenerate across the lesion site up to the medulla. In contrast, the low tide axons stay far behind and fail to regenerate. However, they might recover the terminal clubs (tcs) that remain distanced caudally from the lesion site. The neonatal a-FS feature identifies this watershed with the spot on the assembly line delineating the CP.

The Txs on the upstream assembly line (Figure 2B, High tide) elicit distinguishable features reflecting temporal and spatial aspects of axon development. These findings increase our understanding of axon development and regeneration. On the one hand, the rapid decrease in the number of i-FSs to zero correlates with a temporal element. On the other hand, the axons extending beyond the lesion site shorten their lengths, exhibiting spatial differences. A common denominator that might determine both phenomena (Figure 2B) is assigned to thrust (Discussion). The convincing features of reprogramming are confined to the CP. Whether the difference between high- and low-tide i-FSs implicates different phenotypes is questioned (Discussion).

3. Results

3.1. Regeneration Comes to a Halt before Neap Tide

Regenerated axons rendered by Txs during the CP are classified by the rostral i-FSs (Figure 2B). This reprogrammed phenotype demonstrates a particular variability in axon length. At high tide, the axons regenerate across the lesion site as far as the level of the medulla. At low tide, the axons remain distant from the rostral lesion site. The phenotype displays a uniform axon feature that varies in length. The variability suggests a hidden factor that we assign speculatively to thrust exerting energy for elongation. When the overall amount of thrust peaks, axons can regenerate beyond their cutoff level. This effect is linked to Txs of the pioneering axons still on their way to the medulla but before they transition. Recovered from the impact of Tx, ample thrust propels these high tide axons again into the medulla. The “upstream” unharmed TH.0 stage axons accompany them alongside and transition into colls in the gracile nuclei, as shown in Figure 2B at (4)-(6). The downstream Tx renders more low tide axons. The ensuing i-FSs do not regenerate. We postulate that the fiber elongation process will stop when energy runs out. Those shorter lengths at neap tide may likely represent axons of the intermediate subdivision, of which the multi-level Clarke’s nucleus is their target [12]. The data shows that low-tide axons exhibit gradual length-shortening, whereas graduality is less noticeable in high-tide axons. Nevertheless, an occasional i-FS has been found in the rostral medulla.

3.2. Six Bell Ringer Cases Exhibiting All Dynamic Features

The paradigm induces a short perturbation of the intrinsic development plan. In response to half an hour of hypoxia, DRG neurons are reprogrammed. This susceptibility to hypoxia renders a distinctive feature of axons on the upstream cascade. In the next figures, two infographics (A and B) help us understand the development state in the blink of an eye, just like Figures 1C and D. The colored bars illustrate the imaginable development states of the long primary afferent system in the DC at M₀, i.e., just before the Tx. The line graphic visualizes the artist's impression of the Tx's durable impact on the fiber system. The colors refer to the axon stages on the assembly line (Figure 1). The estimated number of parent axons and i-FSs at M₀ determine the case order of Figure 3, Figure 4, Figure 5, Figure 6, Figure 7 and Figure 8 on the red-blue line (Figure 2B). Figure 3, Figure 4, Figure 5, Figure 6 and Figure 7 demonstrate multi-stage configurations, mostly in combination with upstream parent axons. They accomplish the physiological development down the cascade and penetrate the gracile nuclei. Figure 7 shows the last i-FS at a sub medullary level. Figure 8 shows an a-FS outlier exhibiting a single-stage monomorphic configuration at the level of the medulla (Discussion). Finally, Figure 5 exhibits low tide axons with decreased axon lengths, reflecting inferred minimal thrust.

Legend forFigure 3, Figure 4, Figure 5, Figure 6, Figure 7 and Figure 8

*/**	asterisk/s	a single asterisk indicates a TH.1-stage i-FS / a tandem asterisk indicates colls
g.nu	gracile nucleus
4th V	fourth ventricle
	L|Th|C	Lumbar|Thoracic|Cervical segment of the spinal cord
	c <== ==> r	caudal <== ==> rostral direction of the spinal cord
	d <== ==> v	dorsal <== ==> ventral in a sagittal section of the spinal cord
L/R	left side/right side	of the spinal cord in a horizontal section
N	fix pinhole	an artifact from tissue processing; a number identifies the spinal cord's level derived from the gelatine block's count spinal level of Tx / depicted level in the Figure

In the medulla of the T78.p40 male (Figure 3C), the high tide axons exhibited the multistage axon configuration. This finding suggests an upstream M₀, likely occurring during the first half of the CP. The axons downstream of the severed ones, which had transitioned upstream, reflected full thrust and transitioned into the TH.2 colls, i.e., after the Tx. These predominantly labeled the gracile nucleus on the left side. The amount of labeling indicated a reduction in the number of axons in line with a downstream origin. On the right side, the fibers may partially counterbalance the loss observed on the left side. The i-FSs were discernably tipped with blunt tcs at both the right and left gracile nuclei, underlining a fetal Tx. Caudally from the medulla (Figure 3D), one i-FS was visible in each DC, possibly originating from a neuron in the most caudal DRG at L6.

In the medulla of the T42.p240 female (Figure 4C), various TH.2 staged colls were present in the left gracile nucleus. The axon reduction indicated that the M₀ fell downstream compared to the former p40 male. Here, the less distinct hallmark a-FS was age-morphed and exhibited just a few TH.1 stage i-FSs abutting the caudal lesion site. A dorsal surface tag marked the lesion site (Figure 5D). That might be formed by external scar tissue. This stigma covered the lesion site's CG glia, different from the outmoded term glial scar [25][2301].

After almost two years, the left gracile nucleus of the V1.p600 male (Figure 5C) exhibited numerous regenerated i-FSs. A few colls might be present too. In contrast, the i-FSs of low tide fibers distanced from the lesion site indicated the inability to regenerate beyond the lesion site. However, these i-FSs contained labeling indicating that they had recovered. The shorter lengths were assigned to insufficient thrust at M₀ beyond the closure of the CP in these low tide axons (Figure 2B). The axons were distributed throughout the caudal DC. The reduction in lengths was paramount and permanent. After almost two years, these i-FSs tipped with the typical blunt terminal clubs (tcs) were still distinguishable from the parent axons. Moreover, they were found on either side of the lesion site, i.e., at caudal and bilateral rostral levels.

The hallmark a-FS of the Tx’s impact in the neonatal W2.p9 male abutted the cervical lesion site at a dorsal hump (Figure 6D). The multimorphic lesion configuration also entailed the TH.0 parent axons. They gave rise to TH.2 colls, which had penetrated the left gracile nucleus. Moreover, various colls had grown into the CG at the lesion site. These features might be linked to the TH.1 axons at the medulla severed just before or while transitioning down the cascade.

The W20.p6 neonate was comparable to the former p9 with similar features (Figure 7). The submedullar DC entailed a single i-FS that had not reached its target at the level of gracile nucleus, devoid of labeling. Again, the three axon development stages were gathered at the lesion site, marked by an intraspinal cyst. A superfluous amount of TH.2 colls scattered throughout the neuropil. This feature of dystopic growth illustrated the paradigm’s impact by elucidating the contrast between this downstream axon sprouting and de facto upstream CNS axon regeneration features of the high tide rostral i-FSs.

The medulla of the W14.p14 female (Figure 8C) showed surfaced axons in the DC bypassing the gracile nucleus. Their continuity with the bundled i-FSs fanning out beyond the ventral gracile nucleus was taken for granted. The appearance resembled the hallmark a-FS while morphing by age. The i-FSs looked like they had grown far beyond their proper ventral targets. Notably, the colls were absent in the gracile nucleus. This peculiar single-stage, monomorphic configuration in the upper medulla raises questions about what mechanism might be accountable for the outlier feature (Discussion). The one-of-a-kind lesion site exhibited a multilevel diastematomyelia, masking the exact Tx level. The pathology might have been irrelevant for the i-FSs bypassing in the left DC. The most upstream cut TH.1 & TH.2 axons (Figure 8A) terminated caudally into the lesion site (not shown).

4. Discussion

Pioneering long primary afferent axons regenerate toward the gracile nucleus if cut during the CP. The pertinent features during this narrow time window have been identified in a previous study [12]. Half an hour of hypoxia induces reprogramming of the neuron’s phenotype. This permanent axon feature is distinguishable over time by its blunt fiber tip, just like the well-known terminal club. This feature of the afferent axon materializes the CNS regeneration enigma by definition. The i-FSs bear doubtless characteristics of the obligatory terminal clubs, the regrowth after their convincing severance, and the mapping of their natural targets. We want to add a pivotal one that has been overlooked, i.e., the high tempo of axon elongation.

Revealing the conditions of enigmatic regeneration uncovered during the CP of the intrinsic development plan raises questions. Due to susceptibility to less oxygen, the cut axons demonstrate the highest dynamic features. On the one hand, the neuron’s reprogrammed phenotype blocks development further down the cascade. On the other hand, the upstream severed i-FSs elongate across the lesion long-distance. In contrast, the downstream cut axons elongate at variable lengths and remain caudally distant from the lesion site. This variability indicates a development-linked relationship, which we assign to the decline in inferred energy [12].

This paper describes the recovery and long-distance re-elongation of cut high-tide axons extending as far rostrally as the medulla at an inferred high speed. A capability that is assigned to high thrust levels. This uptempo axon elongation might be due to the combination of conditioning by the Tx and hypoxia. Spinal cord transections and hypoxia are known conditions that can lead to elevated ATP levels [26,27]. These phenomena might even show synergy, called hypoxic conditioning. Regarding the paradigm’s physiological pathway, the Tx’s sequelae suggest that situational conditions possibly determine where or when the f-ES and s-ES ultimately occur. Our previous paper addressed the likelihood of a non-prevailing intrinsic development plan regarding the wandering i-FSs' whereabouts in the DC [12]. Nevertheless, the hypothetical s-ES phenomenon in high and low tide axons and the blueprint's TH.2 transition normally coincide in some consecutive manner [28]. Adhering to our blueprint’s concept, this option might also unveil a possible affinity between their neuron’s transcript- and translatomes governing the hypothetical TH.1 transit. So, might the transition be similar in high and low tide axons? Simplifying the pertinent proteomic signatures might ease future rejuvenating strategies backward on the upstream cascade.

A key question remains whether the mature state can rejuvenate and reinstate the conditions of the proper transitions. The data provides evidence that a lack of thrust can be accountable for the system’s failure to recapture. The history of negative results implies a catch-22. Low thrust might limit the impact of artificial tools such as cell cultures and organoids. Referring to our blueprint, we question the adequacy of equating axon sprouting or time-consuming long-distance axonal projection with long-distance axon elongation. Only the upstream i-FS with bluntly tipped tcs demonstrate regeneration beyond the lesion site.

Our data may comply with a simplified blueprint, accustoming the TH.1 hub. The lengths of the high tide i-FSs and a-FSs differ from those of low tide i-FSs; they all might demonstrate merely thrust-driven effects in contrast with primarily cell cycle dependency [29]. We postulated in our previous paper that the hypoxic conditions during spinal cord lesions might affect the inferred speed of axon growth, reflecting the variability of the low tide i-FS lengths, in particular [12]. Again, the growth speed relationship between axon length and inferred thrust has gained credibility. The speed slows in low-tide axons but might also gear up in high-tide axons at spring tide. An uptempo of speed might explain why the a-FS bypasses its medullary target (Figure 8C). Purinergic signaling compound levels can multiply manifold compared to basic conditions [22,30]. Increased energy supply might explain why the pioneering growth cones have bypassed their target at spring tide (Figure 8C, a-FS). This phenomenon indicates thrust is a manipulable commodity, underpinning its situational impact. It is worth noting that half an hour of hypoxia was an estimation of the procedure’s mean duration. Variations in the thirty-minute hypoxia might have a relevant yet undetermined impact on the i-FSs, explaining an increased elongation tempo and farther reach (Figure 8C). The susceptibility to hypoxia is confined to the CP. Beyond that short period, the development in the upstream unharmed axons along physiological pathways is normal. The transient involvement of oxygen-sensing mechanisms potentially points to HIF-alpha signaling pathways [21,22,23,31]. The mechanism permanently affected the cut fibers, some of which regenerated, too. It is questionable whether transcriptome profiling might discriminate upstream from downstream i-FSs [32]. Reference validation for pioneering axons versus coll formation requires reliable profiling tools with development stage specificity. Our data casts doubt on the assumed validity of proteomic profiles of cultured embryonic stem cells without being assessed, as they might have been reprogrammed and exhibit downstream profiles of differentiated TH.2 stage cells [33]. Underestimating the value of evaluated and validated long-distance regeneration profiles might have attributed to the translational complex being overlooked [34]. Qualifying parent axons and classifying colls as proof of CNS regeneration are comparable conundrums for misinterpretion of CNS regeneration [19]. This research may benefit from adding the tempo topic to those CNS regeneration criteria established twenty years ago [19].

The bluntly tipped i-FSs, with their regenerative capacity, illustrate the dynamic features of the developing primary afferent axons in the spinal cord. Profiling of all the omics may help identify the physiological pathways involved in CNS regeneration. The inferred high elongation speed of 1 mm/hour is also plausibly proven by negative demonstration. The lumbar pioneering afferents reach the gracile nuclei at a distance of about 10 mm in (less than) a day. In contrast, published cell profiles, which create colls crossing a few mms after days or weeks, point at the downstream stage in the cascade. The commonly held belief about axon growth speed in the CNS is based on biased experiments [35]. In vivo live-cell imaging of primary afferent axon outgrowth rates at 1 or 2 mm/day [36]. However, whether cultured neuronal progenitor cells whose axons grow slowly manifest the upstream phenotype is questionable. Nevertheless, the widely adhered-to notion of 1 mm/day is considered a rock-solid dogma of axon outgrowth [19]. A 40 µm/hour tempo was recently recorded in organoids, reflecting a similar phenotypical proteomic signature [37].

Growth cone assembly and propulsion are the forefront features of pioneering axons that determine the elongation speed, revealing a manipulable tempo [5,38]. Our data fuels the suggestion of excessive speed, demanding an extreme energy supply. If M₀ falls on the right spot on the assembly line, severing axons and hypoxia might synergistically increase the pool of compounds for purinergic signaling [39]. Understanding ubiquitous purines and associated pathways related to energy supply may lead to pertinent gaps in knowledge, providing opportunities for future research [30]. We sincerely acknowledge the scientific contributions of Sir Geoffrey Burnstock (1929-2020), particularly his influential paper on purinergic nerves [40]. Purinergic signaling provides a potential mechanism for delivering significant amounts of adenosine compounds to the developing axon. It took twenty years to overcome skepticism about Burnstock's purinergic hypothesis [41]. Today, it may be easier to decipher and quantify the compounds of the purinergic signaling system and assess the oldest evolutionary preserved purine receptors required for CNS regeneration [42]. We need to determine whether the proper amounts of pertinent molecules might have an impact, supporting the view of the right molecule at the correct location at the right time [43]. Temporospatially pinpointed experimentation under distinct hypoxic conditions may reveal whether rejuvenation can alleviate preexisting development blocked after reprogramming. While knockout mutants have great scientific value, their single-cell sequencing might reveal randomness and nonphysiological phenotypes. The reprogrammed i-FS’s transcriptome may entail a physiological pathway for research into regeneration. The next step is to determine a possibility for manipulation recapturing an upstream transcriptomic signature with the ability to regrow long-distance axons quickly and form coils during maturation. Cracking these codes is crucial for engineering regenerative conditions in the adult spinal cord despite a repellent extracellular environment.

Our findings show the ability of long primary afferent axons to regenerate, manifesting the i-FS axon feature. The i-FS and the a-FS highlight the dependency on conceptual thrust and manifest the long primary afferent system's perturbed temporal and spatial development blockade. These features also delineate the susceptibility to reprogramming due to hypoxia in the upstream development. Half an hour of hypoxia can be adequate to reprogram this enduring new phenotype. The two significant characteristics may be key to understanding regeneration. Regeneration can occur in the pioneering axons upstream during the spring tide but not in low tide downstream axons, which target Clarke’s nucleus [12]. This discovery may be useful for identifying profile benchmarks for organoids and cell cultures applied in preclinical studies and therapeutic strategies for SCI patients. Profiles reprogrammed physiologically might help to decipher why assumed long-distance regeneration potential in translational research remains ineffective. The assumption is made that those indispensable compounds in purinergic signaling provide the necessary energy for long-distance regeneration, and their discovery revolutionizing preclinical research is expected shortly. Then, the therapies’ potential for clinical outcomes promises a better outlook for SCI patients.

Author contributions: Conceptualization, Frits de Beer; Investigation, Frits de Beer; Resources, Frits de Beer; Visualization, Frits de Beer; Writing-original draft, Frits de Beer; Supervision, Harry Steinbusch FB began this work during the last few months of his residency. He was a qualified neurosurgeon responsible for research, development, and data acquisition over ten years. After retiring, a dormant fascination ignited a second career as a neuroscientist. The internet and open-access literature provided the opportunity to explore an unknown world filled with incomprehensible random features related to basic biology and the conditions of enigmatic CNS regeneration. The fruitful collaboration with HS was tremendously helpful in making the results editable for public access.