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Abstract
Introduction This research endeavors to investigate the phenomenon of intraneural spread across distinct locations: subcircumneurium, extrafascicular intraneural, intrafascicular intraneural, and intraperineurium after deliberate intraneural injections across five mammalian species. The study also aims to propose determinants influencing this spread. Furthermore, the investigation strives to ascertain the optimal animal species and needle configuration for extrapolating intraneural injection outcomes to human contexts.
Methods This study examined 60 sciatic nerves from 30 fresh and untreated cadavers of rats, rabbits, dogs, pigs, and sheep. The specimens were organized into five groups, each comprising an equal number of nerves. Histological assessments were performed on 30 nerves, involving fascicle metrics. The remaining 30 nerves underwent intentional intraneural injections, facilitated by 19G and 23G needles under ultrasound and direct visualization guidance.
Heparinized erythrocytes combined with a methylene blue solution were used as a marker to analyze the extent and patterns of intraneural spread. Needle orifice measurements were obtained, and these data were overlaid onto images of both nerves and needles. This enabled a comparative evaluation of sizes and an assessment of marker diffusion.
Results The findings indicated that sciatic nerves in rats, rabbits, and dogs were oligofascicular, characterized by larger fascicles, whereas pigs and sheep exhibited polyfascicular nerves comprised of numerous smaller fascicles. Fascicular diameters were variable across species, with dogs presenting the largest measurements. While intraneural spread was observed and documented, intrafascicular marker spreading was rare, occurring only in one rabbit specimen. Needle orifice attributes were scrutinized and visually depicted.
Conclusions Despite the formidable challenges associated with the practical realization of intrafascicular injection, the utilization of animal models possessing monofascicular or oligofascicular nerves, such as rats, rabbits, and dogs, in conjunction with needles featuring aperture dimensions surpassing those of the fascicles, likely contributes to the compromised reliability of investigations into intraneural injection outcomes.
- peripheral nerve injuries
- anesthesia, conduction
- nerve block
Data availability statement
All data relevant to the study are included in the article or uploaded as online supplemental information.
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WHAT IS ALREADY KNOWN ON THIS TOPIC
Previous research has used various animal models to investigate subcircumneural, subepineural, and subperineurium injections, collectively referred to as intraneural injections, which have traditionally been blamed for nerve injury.
WHAT THIS STUDY ADDS
Not all animal models are suitable for studying intraneural injections, as observed by the significant variations in the microscopic structures of the nerve elements.
It is essential to select the appropriate animal species to study the spread of injectate in each microscopic compartment of the nerve. Additionally, choosing the correct needle type and size is essential for studying injectate spread within different internal nerve structures.
To ensure the relevance of data translation to human nerves, it is critically important to consider the appropriate relationship between needle size and type, animal species, and nerve type within that species. This study focused explicitly on intrafascicular intraneural injection.
Conclusions drawn from studies using one animal species, including humans, and needle specificity (type and size) cannot be readily extrapolated to another species.
HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY
The validity of conclusions in previous studies on intrafascicular (subperineurium) injection is doubtful and will require re-evaluation.
Previous recommendations based on animal studies have lost their validity and need to be re-evaluated.
Introduction
In addition to direct needle-induced trauma to nerve fascicles,1 2 recent investigations have cast doubt on the plausibility of nerve injury resulting from intraneural injection with subsequent intrafascicular spread, suggesting a high degree of improbability or even impossibility.3 4 For more than five decades, the concept of intraneural injection has been dichotomized into intrafascicular (unsafe) or extrafascicular (safe) categories, largely disregarding the distinct neural compartments.5 6 This underscores the necessity to ascertain and corroborate, across diverse animal species, the potential regions of spreading subsequently to purposeful intraneural injection.
To avert terminology confusion, it is imperative to comprehend the synonyms for various terms: extraepineural (extraepineurium) corresponds to subcircumneural; extrafascicular intraneural aligns with subepineural (subepineurium); intrafascicular intraneural equates to subperineural (subperineurium); and intraperineural (intraperineurium) signifies the interstitial space between perineurium cell layers. Furthermore, the ability of intentional intrafascicular injection and its subsequent spreading has recently been questioned in human subjects.3 The perception that such injections may induce axonal damage7 8 has resulted in a notable bias against intraneural injection, culminating in the design of disposable manometers to monitor injection pressure during peripheral nerve block administration, thereby allegedly mitigating the risk.9–14 However, before any conclusions can be drawn, the plausibility of intrafascicular injection occurrences in animal models must be established. Conversely, it is worth noting that extrafascicular intraneural injections do not yield electromyographic changes indicative of nerve injury, as evaluated over a 6-month span.6 15 16 Some even posit that minimal-dose subepineural injections (extrafascicular intraneural) might be advantageous, yielding optimal nerve blocks in terms of onset time and duration of anesthesia.17
Much, if not the entirety, of the supportive evidence for the detrimental implications of intrafascicular injection has been extrapolated from studies involving animals.7 8 18–20
Given the conflicting evidence and the necessity to scrutinize the mechanisms underlying intraneural spread within specific animal species, thereby establishing a dependable animal model whose findings can be extrapolated to human scenarios, our primary objectives encompass evaluating the potential spread locations (subcircumneural, subepineural, subperineural, or intraperineural) following deliberate intraneural injections across five mammalian species. Additionally, we sought to confirm the plausibility of intrafascicular intraneural injections, while our secondary aim was to determine the most suitable animal species and optimal needle size/type combinations that hold promise for extrapolating outcomes of intrafascicular or extrafascicular injections to human contexts.
Methods
Light-microscopic images were captured and analyzed from the sciatic nerves of fresh animal cadavers in five different groups: group 1 consisted of six Wistar rats, group 2 included six New Zealand rabbits, group 3 involved six Beagle dogs, group 4 comprised six hybrid pigs, and group 5 consisted of six Ripollesa sheep (table 1).
Diameter of the fascicles (primary and secondary) in the five animal species
The study was conducted at the Vall d'Hebron Research Institute in Barcelona, Spain. The histological examinations were performed at the Institute of Applied Molecular Medicine, School of Medicine, University of CEU San Pablo in Madrid, Spain. The animals were handled according to internationally accepted ethical principles of the 3Rs, which encompass replacement, reduction, and refinement.
The methodology encompassed four distinct components: (A) a morphological investigation aimed at discerning interspecies variations, facilitating the identification of analogous neural structures to those present in humans; (B) deliberate intraneural injections performed to delineate potential sites of extraneural and intraneural spreading across all species (extrafascicular, intrafascicular, intraperineural); (C) an examination of needle characteristics, which involved documenting the attributes of the needles used in the present study as well as considering alternative needle options for subsequent research endeavors; and (D) an evaluation of fascicle dimensions and needle orifice sizes employed in this investigation, intended to elucidate the underlying factors contributing to the observed outcomes.
Tissue preparation and morphological study
To start the morphological study, 60 sciatic nerve dissections were performed bilaterally from each animal (n=30 animals) immediately after being euthanized. Half of the sciatic nerves (30) were used for morphological studies—the uninjected nerves. The other half (30)—the injected nerves, were injected with a marker (see marker details and injection methods below). The sciatic nerves were dissected from the posterior compartment of the leg, proximal to their division into the tibial and common fibular (common peroneal) nerves. The dissections involved meticulously removing excess fat and connective tissue surrounding the nerves, maintaining their anatomical integrity.
Histological analysis of the uninjected nerves
Two 2–3 mm long samples were obtained from each uninjected sciatic nerve at intervals of 5 mm in smaller animals (rats and rabbits) and 10 mm in the larger animals (dogs, pigs, and sheep)—a total of 60 pieces from 30 nerves. The transverse cross-sectional pieces of the nerves were fixed in 10% buffered formaldehyde for 30 days. The tissue pieces were subsequently processed with paraffin wax, and microtome-assisted sectioning at 20 micron intervals (3 µm in thickness) resulted in 10 successive slices. Staining with H&E was performed on all the sections under standard conditions. Light microscopy was examined at an initial ×800 magnification using a Leica DM5500 B microscope (Leica SCN Microsystems Wetzlar, Germany). The captured images were documented using a Leica DFC425 camera and saved using a Leica SCN400 Slide Scanner (Meyer Instruments, Houston, Texas, USA).
Image analysis of the histological sections was done using ImageJ software (National Institutes of Health, Bethesda, Maryland, USA).
The primary and secondary diameters of all fascicles were quantified within the cross-sections. The primary fascicular diameter was defined as the imaginary line crossing the fascicle in the same direction as the needle’s advancement during nerve penetration (as an anteroposterior approach at a 90° to the nerve axis—type 1 approach). The secondary fascicular diameter was defined as the imaginary line crossing the fascicle at a 90° angle from the primary diameter (as a latero-medial approach to the nerve axis—type 2 approach).
These measurements were necessary to assess the compatibility of the needle diameters (see below).
Deliberated intraneural injections and histological analysis
Preparing the injected nerves in a similar way as the uninjected nerves, the histological analysis involved evaluating extraneural marker spread (outside the circumneurium or epineurium), intraneural extrafascicular marker spread (outside the perineurium), intraneural intrafascicular spreading (inside the perineurium), and spreading within the thickness of the perineurium between the cell layers of the perineurium; spreading type previously described by Reina et al.3
To assess the spread of the marker inside or outside the fascicles in each species, intraneural injections were performed using heparinized erythrocytes as the marker.21 The marker consisted of human blood obtained from the aspirations from the surgical fields of scheduled spinal surgeries at the Vall d'Hebron Orthopedic Surgical Hospital from patients who did not have any infectious or hematological pathology.
The patient’s hematocrit was between 32% and 40%, and for each 25 mL of whole blood, 25 000 IU of heparin was added and diluted with 0.9% saline to a hematocrit of 15%. Blood samples from different patients were not mixed. The final marker was a mixture of the erythrocytes described above and a methylene blue solution. This solution was prepared by keeping a ratio of 1 mL of methylene blue to 50 mL of a heparinized erythrocyte solution.21
Because the erythrocytes remained within the perineurium compartment into which the solution had been injected, the erythrocyte-heparin-saline-methylene blue mixture was selected to replace the traditional use of India ink to achieve better results.22 Furthermore, as previously argued, if only methylene blue were used, it would be removed from histological images during the washing process with solvents used during histological preparation.
Before the injections, the sciatic nerves were identified under ultrasound guidance using a Mindray M7 ultrasound machine (Shenzhen Mindray BioMedical Electronics, Nanshan, Shenzhen, China). A linear transducer L14-6Ns, operating at 8–12 MHz, was used for the smaller animals (rats, rabbits, and dogs), while a convex transducer, C5-2s, operating at 2.5–5 MHz, was used for pigs and sheep. Ultrasound images were acquired by placing the scanner between the ischial tuberosity and the greater trochanter of the femur and following the nerve distally. The sciatic nerve’s bifurcation into its two branches, the tibial nerve and the common fibular (common peroneal) nerve, was thus identified. Subsequently, minimal dissections were performed to expose the external surfaces of the sciatic nerves while retaining the nerves within their surrounding tissues.
Then, under direct vision and without further ultrasound guidance, the neural punctures and intraneural injections were performed in ‘types 1 and 2 approaches’ toward the nerve and fascicles, visualizing the widening of the nerve and introducing a marker solution that altered the color of the nerve (methylene blue solution). Second, ultrasound images were compared with the initial images to verify the swelling effect. Due to their small size, the sciatic nerves of rats and rabbits were punctured with 23G needles (Locoplex, bevel angle 17°, length 35 mm, Vygon, Paterna, Valencia, Spain). In contrast, the sciatic nerves of dogs, pigs, and sheep were punctured with 19G needles (Stimulong NanoLine, Facet tip, length 50 mm, Pajunk, Geisingen, Germany).
The injected marker volume also varied between species, with rats receiving 0.5 mL, rabbits receiving 1 mL, and dogs receiving 2 mL. Pigs and sheep nerves were injected with 5 mL.
Analysis of needles
This examination was conducted on various needles with different diameters and tip designs, including the needles used for this study. Measurements of the length and width of the needle orifice ellipses were taken using a stereoscopic microscope camera (Leica 56D microscope and Leica EC3 camera, Leica SCN Microsystems).
Evaluation of the relative fascicle dimensions and needle orifice sizes
Image Processing with Photoshop Microscopic images of nerves and needles, captured at the same magnification, were superimposed to facilitate the interpretation of the findings following intraneural injections. Adobe Photoshop software (CC 2017.01 version, Adobe System, San Jose, California, USA) was used to determine whether the partial or entire needle orifice could fit and thus be inserted into a fascicle.
Measurements
The principal metrics of this study were the primary and secondary diameters of the fascicle, as defined above, and the lengths of the needle orifices. The subsequent metrics measured the size of the needle orifices for a series of needles. The third observation involved evaluating the spread of the intraneural injection of a marker and identifying its presence inside and outside the nerve, as well as inside and outside the fascicles.
Statistical methods
Twelve sciatic nerve samples were considered for each species to conduct statistical analyses. The variables compared in each of the groups fulfilled the properties of the normal distribution according to the Kolmogorov-Smirnov Z-test. Data were expressed using mean, SD, 95% CI, maximum value and quartiles. Group comparisons were performed using the Kruskal-Wallis test (H test). Post hoc pairwise comparisons were conducted using Mann-Whitney U exact tests due to the small sample sizes. The Bonferroni-corrected significance threshold (p<0.005) was applied for the ten pairwise tests. All statistical analyses were performed using SPSS, Released 2009, PASW Statistics for Windows, V.18.0, SPSS.
Results
No samples were excluded from the analysis. The histological features of the sciatic nerves and the patterns of intraneural distribution are presented in figures 1–5. Measurements of the primary and secondary fascicular diameters, including mean, SD, 95% CI, maximum value, and IQR, are summarized in table 1. Table 2 contains the statistical analysis results comparing the values among different animal species. It evaluates the differences in various parameters. Table 3 provides the dimensions of the orifice of the 23G and 19G needles used in this study, along with measurements of other alternative needles suggested for potential future experiments.
Cross-sectional view of the rat sciatic nerve showing the site of intraneural injection using heparinized erythrocytes as a marker. (1) Heparinized erythrocytes were used as a marker, (2) Axons are located within the fascicles. (A) The tibial and common fibular nerve fascicles are visible, both devoid of the marker. The marker is localized in the adjacent adipose tissue but outside the fascicles. (B) Superimposed images of the rat sciatic nerve and the 23G needle (Locoplex, bevel angle 17°) used for intraneural injection, captured at the same magnification using Adobe Photoshop (Adobe, San Jose, California, USA). The length of the needle opening exceeds the diameter of the fascicle (type 1 approach). The marker did not penetrate inside the fascicle but was drawn into the adipocytes outside the fascicle through the unoccupied area of the needle opening. (C and D): Detailed views of the fascicles reveal the marker’s presence outside the fascicles and the marker-free axons. The perineurium is indicated in blue, while the collagen fibers surrounding each fascicle are represented in green. There is close contact between the perineurium and these collagen fibers. These images were processed using Adobe Photoshop (Adobe, San Jose, California, USA), with the perineurium assigned as blue and the collagen fibers as green for enhanced clarity and comparison. Magnification: original acquisition, A and B: ×200, C and D: ×800. Scale bar: 1 mm.
Cross-sectional view of the rabbit sciatic nerve illustrating the location of an intrafascicular injection, the sole instance of intrafascicular spread detected. The injection involved the use of heparinized erythrocytes as a marker. Heparinized erythrocytes served as the marker, (2) Axons are within the fascicles. (A) Accumulation of marker particles is observed within the tibial nerve fascicle, while the common fibular nerve fascicle remained free of the tracer. The tracer was also identified within the adipose tissue between the neighboring fascicles, but outside of them. (B) Two images were acquired from the rabbit sciatic nerve and the 23G needle (Locoplex, bevel angle 17°) used for the intraneural injection. These images, captured at the same magnification, were superimposed using Adobe Photoshop (Adobe, San Jose, California, USA). The length of the needle orifice is visibly equal to the diameter of the fascicle (type 1 approach). The marker entered the fascicle. (C) Detailed view of the fascicle exhibiting spreading of the marker amidst the layers of the perineurium (blue). Surrounding the fascicle is an intricate interlacing of collagen fibers (green). (D) Detailed view of the fascicle displaying the spread of the marker within the fascicle and between the layers of the perineurium (blue). The fascicle is covered by a network of interwoven collagen fibers (green). Magnification: original acquisition, A and B: ×200, C and D: ×800. Scale bar: 1 mm.
Cross-sectional view of the dog sciatic nerve demonstrating the site of an intraneural injection employing heparinized erythrocytes as a marker. Heparinized erythrocytes were used as the marker, (2) Axons are located within the fascicles. (A) Both the tibial nerve fascicle and common fibular nerve fascicle appear devoid of the marker. The marker is localized within the nearby adipose tissue, but outside the fascicles. (B) Three images were acquired from the dog sciatic nerve, depicting the 23G needle (Locoplex, bevel angle 17°) and the 19G needle (Stimulong NanoLine, Facet tip, Pajunk), captured at the same magnification and superimposed using Adobe Photoshop (Adobe, San Jose, California, USA). The 23G needle was positioned in the common fibular fascicle, while the 19G needle was placed in the tibial fascicle. However, the puncture was solely performed using the 19G needle. The length of the needle orifice is nearly equal to the diameter of the tibial fascicle (type 1 approach). The presence of the 23G needle allowed us to assess the potential outcome if that needle had been used. The marker did not enter the fascicle but instead dispersed into the adipocytes outside the fascicle through the unoccupied area of the needle orifice. (C) Detailed view of the fascicle highlighting the presence of the marker outside the fascicles, along with the marker-free axons. The perineurium is indicated in blue, while a network of interwoven collagen fibers surrounding the fascicles is represented in green. Close contact is observed between the perineurium and these collagen fibers. These two images were processed using Adobe Photoshop (Adobe, San Jose, California, USA). Magnification: original acquisition, A and B: ×200, C: ×800. Scale bar: 1 mm.
Cross-sectional view of the pig sciatic nerve illustrating the location of an intraneural injection employing heparinized erythrocytes as a marker. Heparinized erythrocytes served as the marker, (2) Axons are within the fascicles. (A) Both the tibial nerve fascicle and common fibular nerve fascicle are comprised of multiple smaller fascicles and appear devoid of the marker. The marker was found within the adjacent adipose tissue outside and inside the tibial component. (B) Two images were acquired from the porcine sciatic nerve using a 19G needle (Stimulong NanoLine, Facet tip, Pajunk). These images, captured at the same magnification, were superimposed using Adobe Photoshop (Adobe, San Jose, California, USA). The length of the needle orifice exceeds the size of the tibial fascicle (type 1 approach). The marker did not penetrate inside the fascicle but instead receded into the adipocytes located outside the fascicle through the unoccupied area of the needle orifice. (C) Detailed view of the fascicle displaying the presence of the marker outside the fascicle, while the axons remain marker-free. The perineurium is indicated in blue, and a network of interlaced collagen fibers surrounding the fascicles is represented in green. Close contact is observed between the perineurium and these collagen fibers. These two images were processed using Adobe Photoshop (Adobe, San Jose, California, USA). Magnification: original acquisition, A and B: ×200, C: ×800. Scale bar: 1 mm.
Cross-sectional view of the sheep sciatic nerve depicting the site of an intraneural injection utilizing heparinized erythrocytes as a marker. Heparinized erythrocytes were used as the marker, (2) Axons are situated within the fascicles. (A) The tibial nerve fascicle, common fibular nerve component, and all other fascicles appear free of the marker. The marker was localized in the nearby adipose tissue but remained outside the fascicles. (B) Two images were obtained from the ovine sciatic nerve using a 19G needle (Stimulong NanoLine, Facet tip, Pajunk), captured at the same magnification, and superimposed using Adobe Photoshop (Adobe, San Jose, California, USA). It is observed that the needle orifice was larger than the fascicles of the tibial and fibular components (approach type 1). The marker did not enter the interior of the fascicle but instead retracted into the adipocytes outside the fascicle through the unoccupied area of the needle orifice. (C) Detailed view of the fascicle displaying the presence of the marker outside the fascicles, while the axons remain free of the marker. The perineurium is indicated in blue, and a network of interlaced collagen fibers surrounding the fascicles is represented in green. Close contact is observed between the perineurium and these collagen fibers. These two images were processed using Adobe Photoshop (Adobe, San Jose, California, USA). Magnification: original acquisition, A and B: ×200, C: ×800. Scale bar: 1 mm.
Statistical analysis comparing the diameters of fascicles among the five animal species
Needle data proposed as an alternative for future experimental studies, including the needles used in the current paper
Figures 1–5 depict the superimposed images of the needle orifice on the cross-section of the nerve in different species, illustrating the potential positioning of the needle within the nerve fascicles and its possible effects. The corresponding images of all analyzed needles are depicted in figure 6.
Needles: Various needle types are depicted as examples for potential use in future studies, depending on the specific objectives. In this study, needles 1 and 8 were employed. 1: 19G needle (Stimulong NanoLine, Facet tip, Pajunk) 8: 23G needle (Locoplex, bevel angle 17°).
The sciatic nerve appeared macroscopically as a single entity, but microscopic examination revealed the presence of distinct fascicle groups within it. These fascicle groups, which will give rise to nerve branches in more distal regions, are called ‘components’. Notably, the tibial and the common fibular components were identified within the sciatic nerve, comprising groups of fascicles that eventually form these nerves in more distal locations. Two distinct types of sciatic nerves were observed: oligofascicular in rats, rabbits, and dogs and polyfascicular in pigs and sheep.
In the oligofascicular type, the nerves had larger fascicles, representing the tibial and common fibular components. Each component was compact, monofascicular, and mainly integrated by neural tissue. Each was surrounded individually by a thin perineurium closely adhered to a thicker epineurium. Depending on the cross-sectional level of the nerve, smaller fascicles were also identified as components of the sural nerve and muscle branches. Adipocytes were present among these larger fascicles.
The primary and secondary fascicular diameters were consistently larger in the tibial component compared with the common fibular component in all oligofascicular animal specimens. Comparing the primary and secondary diameters of fascicles, the dogs had the largest values, followed by the rabbits and the rats. Statistically significant differences were observed between all analyzed values, except for the fibular component, where the primary diameter of the rat was comparable to that of the rabbit, and the secondary diameter of the rat was similar to that of the sheep. Nevertheless, the rat exhibited larger overall fascicular diameters than pigs and sheep (tables 1 and 2).
In the polyfascicular type observed in pigs and sheep, the tibial and common fibular components were larger than those in rats, rabbits, and dogs. Internally, these components comprised numerous small fascicles separated by adipose tissue. The primary and secondary fascicular diameters in pigs and sheep were smaller than in rats. The primary diameter of fascicles within the tibial component of pigs and sheep was approximately 9 and 7 times smaller, respectively, compared with dogs. In the common fibular component, these values were about 4.6 and 3.6 times smaller than in dogs.
The pig’s tibial component had an average of 39 fascicles (±11.6, 95% CI 31.7 to 46.4), while the fibular component had an average of 21.6 fascicles (±4.3, 95% CI 18.8 to 24.3). The sheep’s tibial component had an average of 56.5 fascicles (±9, 95% CI 50.7 to 62.2), and the fibular component had an average of 35.7 fascicles (±11.9, 95% CI 28.2 to 43.3).
On analyzing each animal specimen, no intraneural marker spread was observed in the rat, rabbit, dog, pig, or sheep samples (figures 1–5) within the tibial or common fibular fascicles. The rat exhibited a sciatic nerve proximal portion with a monofascicular type and a more distal portion with an oligofascicular type, where a single large fascicle was identified as the tibial component of the nerve and another fascicle as the common fibular component of the sciatic nerve.
The marker was found outside the epineurium in rats, rabbits, and dogs; no marker spread within the fascicle was observed except for one rabbit sample, where marker particles were detected between the axons of the tibial component of the sciatic nerve. In rats and rabbits, the same 23G needle type was used, which had an orifice length greater than the primary diameter of the largest fascicle in rats and very close to the primary diameter of the largest fascicle in rabbits.
In dogs (figure 3), which exhibited an oligofascicular profile with two larger fascicles compared with rats and rabbits, no intraneural marker was observed, although a different needle type was used. The puncture was performed using the same 19G needle type with a larger orifice in dogs, pigs, and sheep.
On analyzing the dog samples, erythrocytes were found outside the nerve. In this case, the length of the needle orifice was slightly greater than the primary diameter of the fascicle (figure 3).
In pigs and sheep (figures 4 and 5), a polyfascicular structure was observed, with the sheep’s fascicle being larger than the pig’s fascicle. The size of the whole sciatic nerve in these specimens allowed for using a needle with a large orifice, like those used in human clinical practice. The advantage of using such a needle was that its orifice length could encompass entire fascicles (tissue with high resistance to liquid entry) and the adipose tissue between them, which has a low resistance to liquid spreading. As a result, it was easy to identify an intraneural marker in all polyfascicular samples, with the marker spreading outside the fascicles (intraneural but extrafascicular). In these samples (pig and sheep), all the fascicles were free of the marker, and the axons did not come into direct contact with the marker in any of the samples.
Finally, in all samples, there was spreading of the marker through the thickness of the perineurium, encountering marker particles among the layers of the perineurium.
Discussion
The meticulous selection of the needle and the animal species was pivotal in determining the incidence of intrafascicular intraneural injections. Hence, it is imperative to acknowledge that not all animal models universally apply to diverse research objectives, potentially yielding varied outcomes of intraneural injections. Notably, the dimensions of fascicles,20 such as those found in the sciatic nerves of rats, rabbits, and dogs categorized as oligofascicular nerves, distinctly differ from the polyfascicular sciatic nerves of pigs and sheep, where multiple smaller fascicles are present. Consequently, the repercussions after intraneural injections are also anticipated to diverge.
Interestingly, an injection beneath the epineurium within an oligofascicular nerve essentially constitutes an intrafascicular injection since the epineurium and perineurium, in such instances, are closely juxtaposed without the interposition of adipose tissue. The juxtaposition of needle orifice and fascicle in composite images offered valuable insights into the mechanics that impede the propagation of intrafascicular spreading following a calculated intraneural injection. We have identified a shared modality that inhibits intrafascicular injections, where the sizes of both the needle orifice and the fascicle serve as pivotal determinants. Our current animal-based investigation corroborates the preceding outcomes.3
Intrafascicular injection is attainable only if two criteria are concurrently fulfilled: first, the length of the needle orifice equates to or is less than the diameter of the fascicle; second, the orifice remains completely nestled within the confines of the fascicle. In practice, accomplishing intrafascicular injection is nearly unattainable unless a suitably proportioned needle is chosen. The gage of the needle orifice plays a pivotal role in ascertaining whether the marker substance will disseminate within or beyond the fascicles. Most samples across diverse animal types exhibited no intrafascicular spreading; this phenomenon was only observed in a solitary instance, specifically in a rabbit nerve.
The interplay between the needle orifice’s size and the fascicle’s dimensions demands careful consideration for each distinct animal model. Deploying the same needle across different animal models yields variable intrafascicular or extrafascicular distribution outcomes. When extrapolating research findings from animal models to human scenarios, a comprehensive understanding of fascicle size, morphology, and intraneural adipose tissue is imperative. This necessitates the selection of a needle with an orifice size commensurate with human fascicles, akin to those used in clinical settings. Beyond the needles used in our current study, supplementary needle options were assessed in figure 6, encompassing orifice measurements (as presented in table 3). While these needles were not initially designed for peripheral nerve blocks, they can be considered for experimental purposes if required. Figure 6 notably illustrates the influence of needle type and bevel angle on orifice dimensions. This aspect has often been overlooked, and needles from disparate manufacturers may encompass differing orifice dimensions despite possessing identical external diameters.
An intriguing query arises regarding the prevalence of extrafascicular marker spreading instead of intrafascicular dissemination. A substantial number of intraneural injection attempts evidenced partial insertion of the needle orifice within the fascicle, resulting in fluid efflux from the fascicles due to the notable resistance offered by the endoneurium.3 This phenomenon holds for most human peripheral nerves, including lateral and terminal nerve plexus branches. Employing a 22G needle with a 15° bevel renders intrafascicular injection exceedingly implausible. Conversely, McLeod et al 23 demonstrated the arduous nature of deliberate intrafascicular intraneural punctures in an in vivo animal model through dynamic video studies, highlighting the impediments of fascicle rotation.
In our present investigation, a 23G needle with a 17° bevel was employed in the sciatic nerves of rats and rabbits, necessitated by the limited diameter of the entire nerve, thus precluding using a larger-gage needle. In the case of dogs, pigs, and sheep, a 19G needle was used, aligning with the nerve size in these animals.
Remarkably, none of our study specimens exhibited marker cells (erythrocytes) within the fascicles, barring a singular instance in a rabbit sample where the marker was detected in a tibial component fascicle among the axons. Within the rat, the tibial component consisted of a single larger fascicle and a smaller one for the common fibular component. However, the 23G- needle’s orifice size slightly exceeded that of the tibial fascicle, precluding complete encapsulation of the orifice within the fascicle. Consequently, all erythrocytes were located external to the nerve. There were no intraneural adipocytes present. In rabbits, the orifice length (23G needle with a 17° bevel) closely approximated the diameter of the tibial fascicles, and intrafascicular spread occurred solely in one instance. This occurrence might have emerged due to fulfillment of the first criterion across all rabbit nerves, while simultaneous fulfillment of both criteria led to intraneural injection in a singular case.
Laredo et al 24 explored intraneural injections using a 25G needle in an in vivo rabbit model under direct visualization, inserting the needle along the sciatic nerve’s longitudinal axis at an angle of around 30°. Their findings indicated intrafascicular lesions in a single instance out of 32 samples. Histological alterations postinjections were generally mild in rabbits, with no discernible impairment to motor, proprioceptive, or sensory functions observed during their study period.
Although our study adopted a 23G needle (figure 6 (Number 8)) with a larger external diameter than Laredo’s 25G needle (figure 6 (Number 12)), the orifice length was intriguingly smaller. This underscores the imperative nature of thorough needle analysis for each research endeavor. In our study, a 19G needle with a longer orifice length was used in dogs. Intrafascicular injection was not observed despite the equivalence between the tibial fascicle and needle orifice dimensions. Intrafascicular injection might have occurred if the orifice had been wholly enclosed within the fascicle, though this was not observed in practice. This divergence could be attributed to the fact that the orifice never remained entirely within the fascicle during intraneural injection attempts, failing to fulfill the second criterion. The most likely rationale is that a section of the orifice consistently extended beyond the fascicle, facilitating extrafascicular marker spreading through the loosely arranged adipose tissue, logically absent within the fascicles. This situation could change with a needle possessing a smaller orifice, augmenting the likelihood of intrafascicular injection.
Despite the larger size of their sciatic nerves in pigs and sheep compared with dogs, their polyfascicular pattern engenders numerous smaller fascicles, each ensheathed by intraneural adipose tissue. As previously discussed, the substantial orifice size facilitates interaction with entire fascicles enveloped by fat, creating an anatomical resemblance to human sciatic nerves. Confronted with endoneurial resistance within the fascicles, the fluid preferentially migrated toward the intraneural fat, thus preferring extrafascicular distribution. The considerable orifice size in pig and sheep sciatic nerves effectively precludes intrafascicular injection into the tibial component, akin to human observations. Neither the first nor the second criterion was met in pigs and sheep, implying that using a 22G, 15° needle in human nerves would yield similar outcomes.
Conclusively, an unrecognized mode of intraneural injection propagation was demonstrated, characterized by a novel marker’s spreading through the perineurium’s cellular layers, traversing its thickness. This event was initially misconstrued as sub-perineural (intrafascicular) spreading when employing India ink,25 although this led to the widening and separation of perineurial cellular strata.
Several limitations warrant consideration in this study. While our investigation was ex vivo, the commencement of the study immediately postanimal demise aimed to circumvent alterations in nerve elasticity that could influence needle penetration and solution diffusion.
Studies featuring histological depictions of diverse human nerves26–29 will facilitate researchers in identifying the relevant nerve for their investigations, enabling the determination of fascicle maps (topograms). This step will guide researchers in selecting the optimal model for their study.
Clinicians stand to gain from recognizing that an excessively large needle orifice relative to nerve fascicle size renders intrafascicular intraneural injection nearly unattainable. To sum up, our investigation underscores that the absence of markers within fascicles across all samples correlates with a needle orifice surpassing the fascicle’s diameter. However, one instance revealed intrafascicular spreading in a rabbit, where the needle orifice length aligned with or slightly exceeded the fascicle diameter. These findings underscore the importance of choosing appropriate animal models and needles for research.
Despite the formidable challenges associated with the practical realization of intrafascicular injection, the utilization of animal models possessing monofascicular or oligofascicular nerves, such as rats, rabbits, and dogs, in conjunction with relatively small fascicles (rats and rabbits) and needles featuring aperture dimensions surpassing those of the fascicles, likely contributes to the compromised reliability of investigations into intraneural injection outcomes.
Data availability statement
All data relevant to the study are included in the article or uploaded as online supplemental information.
Ethics statements
Patient consent for publication
Ethics approval
This study involves human participants and was approved by Human Ethics Committee of the Vall d’Hebron Research Institute-protocol codes 66/15 and 53/18. Participants gave informed consent to participate in the study before taking part.
Acknowledgments
The authors sincerely thank Natasha Beukes, Ph.D. of Lumina Health, for her assistance with the preparation of the manuscript. They would also like to thank Javier Moratinos-Delgado, technician, Virginia García-García, technician, and Paloma Fernández, Ph.D., of the CEU San Pablo University School of Medicine, Madrid, Spain, for their assistance in the histology images.
References
Footnotes
X @marcosperez_vh
Correction notice This article has been corrected since it published Online First. The title has been amended.
Contributors AS: project development, data collection and management, data analysis, and manuscript writing. AP-B: data collection and data analysis, manuscript writing, and editing. MP-C: data collection and data analysis. ME-C: data collection and data analysis. FL: data collection and data analysis. MAR: project development, data collection and management, data analysis, manuscript writing and guarantor.
Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.