Gross and Histologic Evaluation of 5 Suture Materials in the Skin and Subcutaneous Tissue of the California Sea Hare (Aplysia californica)

Abstract

Invertebrates are increasing in their importance to both the public and private aquarium trade and play a vital role in biomedical research. Surgical techniques have become an important approach to obtaining data and maintaining good health in both of these areas. However, studies examining tissue reaction to suture material in invertebrates are lacking. The current study evaluated the gross and histologic reaction of Aplysia californica to 5 commonly used suture materials, including polydioxanone, black braided silk, polyglactin 910, monofilament nylon, and monofilament poliglecaprone. Histologic samples were graded on the amount of edema (score, 1 to 4), inflammation (1 to 4), and granuloma formation (1 to 4) present, and a final overall histology score (1 to 6) was assigned to each sample. Compared with untreated control tissue, all suture materials caused significantly increased tissue reaction, but the overall histology score did not differ among the suture materials. Silk was the only suture that did not have a significantly increased granuloma score when compared with the control. Although none of the suture materials evaluated seemed clearly superior for use in Aplysia, we recommend silk because of its less robust granuloma induction, which is favorable in a clinical and research setting.

Invertebrates are increasing in their importance in biomedical research and as popular display animals in public and private aquariums. The California sea hare (Aplysia californica) is 1 of the most widely used invertebrate animals in neurophysiology research. Because of its easily accessible ganglia and neurons and greater ease of rearing in captivity, Aplysia has replaced the squid giant axon in much neurophysiology research. Surgical techniques used in Aplysia research include probe placement within various nerves and ganglia.^3,17,18 These techniques require surgical closure of the skin over the probe. An inflammatory reaction around a nerve can alter its excitability and associated research findings.⁵ Therefore, using a suture material that causes minimal inflammation and tissue reaction around the target nerve is prudent. Silk is perhaps the most commonly used suture material in Aplysia research,^14,18,20 even though silk causes some of the greatest tissue reactions in several domestic and nondomestic species.^{11,13,24,26,28}

As the popularity of invertebrates in the aquarium trade grows, an impetus is arising to provide more complete veterinary care for these animals. An increased demand for improved care, the environmental impact of collecting animals from the wild, and the cost of obtaining new animals have led many to pursue treatment of invertebrates over replacement.^9,27 When surgery becomes necessary, suture choice can have a marked effect on incision healing: a suture that causes minimal inflammation and tissue disruption can facilitate the process. To this end, the current study evaluates the gross and histologic reactions of Aplysia to 5 commonly used suture materials: silk, monofilament nylon, polydioxanone, polyglactin 910, and poliglecaprone.

Materials and Methods

Thirteen Aplysia (weight, 93 to 170 g) from a single cohort were obtained from the National Institutes of Health University of Miami National Resource for Aplysia (Miami, FL). The Aplysia were kept in artificial seawater (Crystal Sea Marinemix, Marine Enterprises International, Baltimore, MD) and housed individually in plastic baskets measuring 34.9 × 27.3 × 12.7 cm (Sterilite, Townsend, MA). The baskets floated in a 1500-L circular tank, and air stones were placed in each basket to maintain water circulation through the basket. Additional air stones were placed in the bottom of the larger tank to maintain adequate water flow and dissolved oxygen. Water temperature, salinity, pH, unionized ammonia, nitrite, and nitrate were tested daily, and water changes were performed as needed to maintain adequate water quality. The following parameters were maintained: temperature, 17 to 20 °C; salinity, 34 to 35 ppt; pH, 7.8 to 8.4; unionized ammonia, less than 0.04 mg/L; nitrite, less than 0.2 mg/L; and nitrate, less than 4 mg/L. Red algae (Gracilaria ferox) was fed at approximately 1 g per animal 3 times weekly. The Aplysia were acclimated for 2 d in the experimental tank before use in the study, at which time they showed normal behavior and seldom ‘inked’ when manipulated.

Anesthesia protocol.

Prior to the suture study, 3 concentrations of MgCl₂•6H₂O (Mallinckrodt Baker, Phillipsburg, NJ) were tested for anesthetic efficacy, with 2 Aplysia tested per concentration.²³ The concentrations of MgCl₂•6H₂O were 20 g/L (0.098 M), 50 g/L (0.246 M), and 75 g/L (0.369 M). The corresponding amount of MgCl₂•6H₂O for each concentration was dissolved in 1.5 L fresh filtered water. The Aplysia were placed in individual 3-L plastic containers filled with 1.5 L fresh, aerated tank water, and the anesthetic water was added slowly to the container. Induction time was recorded as the time elapsed from when the anesthetic water was added to the point when there was loss of attachment, loss of righting reflex, no voluntary movement, and no response to stimuli. Once the end point was reached, the Aplysia was removed from the anesthetic bath, rinsed 3 times in fresh tank water, and placed in a new container containing 3 L fresh tank water. Recovery time was determined as time elapsed from placement in the fresh tank water until the animal was alert, able to right itself, responded to stimuli, and had readhered to the tank wall. When the Aplysia were fully recovered, they were returned to their holding baskets. The concentration chosen for the remainder of the study was 50 g/L MgCl₂•6H₂O.

Suture reactions.

This study was conducted in conjunction with a teaching laboratory at North Carolina State University College of Veterinary Medicine (Raleigh, NC). Thirteen veterinary students each conducted the following procedure on a single Aplysia. The Aplysia were placed in individual containers and anesthetized as described. When the animals were fully anesthetized, they were transferred to a towel soaked in anesthetic water and clear plastic wrap was used as a surgical drape. A 60-mL catheter-tip syringe with a 10-French red rubber catheter was used to pass aerated anesthetic water over the ctenidia (gills). A single simple interrupted suture was placed at 1 of 6 sites by using 1 of 5 suture materials; the 6th site was the control site, at which no suture was placed. Tested suture materials were polydioxanone (PDS II, Ethicon, Somerville, NJ), black braided silk (Ethicon), polyglactin 910 (Vicryl, Ethicon), monofilament nylon (Monosof, United States Surgical, Norwalk, CT), and monofilament poliglecaprone (Monofyl, Oasis Medical, Mettawa, IL). All sutures were 3-0 and had a FS1 or C14 (3/8-in. circle, 24 mm) reverse cutting needle to maintain uniform trauma from passage of the needle through the skin. Three sites were located on each side of the animal, approximately 0.5 cm dorsal to the lateral aspect of the foot surface and approximately 1 cm apart. Suture materials were rotated so that all were placed at every location on at least 2 animals. Prior to recovery from anesthesia, a loose single interrupted suture (silk) was used to attach 2 color-coded beads to the caudal aspect of 1 of the parapodia for animal identification. When all sutures were placed, the Aplysia were rinsed 3 times in fresh tank water and returned to their holding baskets to recover from anesthesia.

The animals were monitored daily for behavior, inflammation, healing, and suture loss. At 6 d after surgery, 6 of the 13 Aplysia were anesthetized and the suture and control sites were evaluated; the remaining 7 Aplysia were evaluated 12 d after surgery. After visual evaluation, each site was biopsied (incorporating the suture and surrounding tissues) by using a 6-mm biopsy punch, and the site was closed by using 3-0 silk in a combination cruciate and simple interrupted pattern. The suture was removed from the biopsy, and the tissue sample was placed in Davidson solution for fixation. After 24 h, the tissue samples were transferred to 70% ethanol. For histology, all tissue samples were routinely processed, embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin for evaluation by light microscopy. The tissue biopsies were evaluated by 2 pathologists (ASD, JML) blinded to suture type.

To help derive an overall suture reaction score for each biopsy, 3 semiquantitative subcategories were graded (score, 1 to 4) and then used to calculate a final histology score. The 3 subcategories were edema, inflammation, and granuloma formation. Edema, a common acute reaction in mammalian inflammation, was used here as an indicator of total tissue damage and probably also reflects some loss of osmoregulation due to cell membrane injury in aquatic animals. We defined inflammation as the often loosely arranged cellular inflammatory reaction in Aplysia that lacks the well-defined organization typical of a granuloma seen in reaction to foreign material such as suture. Finally, because Aplysia have different inflammatory cell types than mammals,¹⁶ we defined granulomas as well-organized layers of activated inflammatory cells (amoebocytes), often having central foci of necrosis and accompanied by variable layers of fibroblasts, which expanded and replaced the underlying tissue architecture. An overall histology score ranging from 1 to 6 was determined for each sample: 1, normal tissue; 2, minimal changes—minimal inflammation, edema, and absence of granuloma formation; 3, mild inflammation, edema, and rare, mild granuloma formation; 4, moderate inflammation, edema, and granuloma formation; 5, moderate to severe inflammation, edema, and granuloma formation; and 6, high numbers of inflammatory cells, edema, and large granulomas.

Statistical analysis.

Control scores were subtracted from scores for all sites in each animal. Data were grouped according to suture material and statistically analyzed by using JMP 7 (SAS Institute, Cary, NC). An ordinal logistic model was used to evaluate the effect of individual animal (surgeon variability) and suture material on each factor and the overall histology score. The Wilcoxon rank-sum test was used to compare differences between samples biopsied at 6 and 12 d after suture placement. The Friedman 2-way ANOVA was used to compare reactions among all suture materials, and when significant differences were found, the Wilcoxon matched-pairs signed-rank test was used to determine which pairs differed significantly. A P value of less than 0.05 was regarded as statistically significant.

Results

Anesthesia protocol.

The 2 Aplysia that were placed in 20 g/L (0.098 M) MgCl₂•6H₂O showed an increased activity level with exaggerated movements, relaxation and opening of their parapodia, and increased ‘lipping’ (movement of the tissues around the mouth without exposing the buccal cavity or radulla) within 20 to 30 min, but neither animal became fully anesthetized. The pair of Aplysia placed in 50 g/L (0.246 M) MgCl₂•6H₂O showed an initial increase in movement and activity similar to the first pair but subsequently showed progressive sedation, loss of attachment to the tank, and loss of righting reflex, and after 34 and 35 min, the animals became immobile and unresponsive to stimuli. When these Aplysia were placed in fresh tank water, they became responsive and resumed moving in just seconds to approximately 1.5 min. The pair of Aplysia placed in 75 g/L (0.369 M) MgCl₂•6H₂O became heavily sedated but never fully lost their response to stimuli; in addition, 1 of the 2 animals appeared to shrink. Given the poor reaction of the Aplysia to 75 g/L MgCl₂•6H₂O, higher concentrations were not attempted, and the 50-g/L concentration was used for the remainder of the study.

Suture study.

Although 7 sutures were lost before the animals were sampled, only 2 of those sites could not be identified for biopsy at the time of sample collection. The 5 sites that lost sutures but were still identifiable had reaction scores within the range of the corresponding suture sites and were included in the final analysis. The 2 animals that had completely lost a suture site were excluded from further analysis, bringing the total number of animals used to 11. Because suture materials of each type were lost from 1 or 2 sites, no material appeared to be more prone to loss. When lost sutures were recovered, the surgical knot was intact, so they were not lost due to knot failure.

The ordinal logistic model showed that suture had a significant effect on the degree of inflammation, edema, and granuloma formation (P < 0.0001 to 0.005), whereas the individual animal (surgeon) did not affect the inflammatory score (P = 0.3825) or granuloma score (P = 0.7135) but did affect the edema score (P = 0.0026). Because a Wilcoxon rank-sum test showed no significant differences in overall histology scores between sutures sampled at 6 and 12 d, the data for both days were grouped for the remainder of the analyses. Differences in tissue reaction between control and treatment sites were not grossly evident, but overall histology scores differed significantly (P < 0.0004) among sites (Table 1). Further pairwise comparisons showed that all suture materials had significantly (P = 0.0020 for all) higher overall histology scores than did the control, but these scores did not differ (P = 0.0898 to 1.000) among suture materials. For each suture material, overall histology scores varied widely. Minimal epidermal changes were seen, but variable amounts of edema, inflammation, and granuloma formation were seen within the subcutaneous tissue. The subcutaneous tissue is characterized by moderately loose tissue matrix made up of components that are morphologically similar to mammalian collagen fibrils and skeletal muscle bundles, intermixed with fibroblasts and myofibroblasts (hereafter referred to as ‘subcutis;’ Figure 1). Histologic samples representing mild tissue reaction contained low numbers of amoebocytes infiltrating the subcutis, with minimal or no cellular aggregation (Figure 2 A). Moderate reactions showed an increase in the numbers of amoebocytes throughout the tissue, with larger disruption of the surrounding tissues; in addition, small to moderate granulomas were present (Figure 2 B). Marked tissue reactions were characterized by numerous amoebocytes infiltrating the tissues and large dense granulomas that dramatically disrupted normal tissue architecture (Figure 2 C, D).

Table 1.

Overall histology, edema, inflammation, and granuloma scores associated with the tissue reaction of Aplysia californica(n = 11) to 5 types of suture

	Histology scorea		Edemab		Inflammationb		Granulomab
Suture	Median	Range	Median	Range	Median	Range	Median	Range
Control	1.0	1–2	1.0	1–3	1.0	1	1.0	1
Polydioxanone	4.0	1–6	3.0	1–4	3.0	1–4	2.0	1–4
Silk	2.0	1–5	2.0	1–4	2.0	1–4	1.0	1–4
Polyglactin 910	4.0	1–6	2.0	1–4	2.5	1–4	2.5	1–4
Nylon	4.0	1–6	3.0	1–4	2.0	1–4	2.0	1–4
Poliglecaprone	4.0	1–5	2.0	1–4	2.0	1–4	2.0	1–3

^aMaximum = 6

^bMaximum = 4

Figure 1.

Normal skin biopsy from Aplysia californica. E, epidermis; G, mucus glands; S, subcutis; M, muscle layer. Hematoxylin and eosin stain; bar, 0.1 mm.

Figure 2.

Example tissue reactions to suture materials in skin biopsies from Aplysia californica. (A) Mild tissue reaction (histology score, 2). Relatively loosely arranged inflammatory cells (blue dots are their nuclei) and mild edema (pale areas) focally expand the subcutaneous tissues. Bar, 10.0 μm. (B) Moderate tissue reaction (histology score, 4). The suture tract (asterisk) is surrounded by necrotic debris, well-organized layers of activated inflammatory cells (amoebocytes), and scattered fibroblasts. This granuloma focally expands and replaces the surrounding normal tissue. Bar, 10.0 μm. (C) Severe tissue reaction (histology score, 6) at low magnification. The suture tract (asterisk) is surrounded by a large granuloma. Bar, 0.1 mm. (D) Higher magnification of panel C. Note the more extensive tissue damage and denser layers of activated inflammatory cells surrounding the suture tract. Bar, 10.0 μm. Hematoxylin and eosin stain.

All groups, including the control group, showed large ranges in edema scores. The amount of inflammation differed significantly (P = 0.0049) among sites, and inflammation scores for each suture material were significantly different (P = 0.0020 to 0.0156) from that of the control. However, inflammation did not differ (P = 0.2656 to 1.000) among suture materials. Inflammation scores covered the entire range for all suture types (Table 1). Inflammatory cells were not seen in any of the control samples.

Granuloma scores also differed significantly (P = 0.0314) among sites. All suture materials incited significantly (P = 0.0039 to 0.0156) more granuloma formation than did the control, except for silk (P = 0.2500), which scored intermediately between the control and the other suture materials and differed significantly from none. No significant differences in granuloma formation were found among the different suture materials (P = 0.1367 to 0.8047). Granuloma formation was not seen in any of the control samples but was associated with all suture materials to varying degrees. The observed granulomas were characterized by a densely packed focus of inflammatory cells intermixed with necrotic debris and scattered fibroblasts.

Most of the sutures used to close the biopsy sites after sample collection were lost within 1 wk after placement. The sutures that were recovered had intact surgical knots and were believed to have been expelled or had pulled free due to tension.

Discussion

Black braided silk is an organic, multifilament, nonabsorbable suture material that is produced by silkworms (Bombyx mori). Marked tissue reactions to silk suture have been observed in several species, both aquatic and terrestrial.^{11,13,24,26,28} The tissue reactivity has been attributed both to the material itself (chemical composition) and to its physical properties (multifilament). The multifilament structure of a suture increases the surface area for bacterial colonization and allows for more bacteria to be wicked into the suture tract. Due to this natural wicking of the multifilament sutures, poor performance in aquatic animals would be expected. This expectation is justified in that silk is moderately reactive in koi carp (Cyprinus carpio)¹¹ and incites marked tissue reaction in rainbow trout (Oncorhynchus mykiss)²⁶ and African clawed frogs (Xenopus laevis).²⁴ We anticipated similar results in Aplysia. However, in the current study, silk did not differ significantly from any suture material in edema or inflammation score and yielded a median histology score of 2.0 compared with an overall median score of 4.0 for the other suture materials; this difference was not significant due to the high variability in scores for all sutures. However, silk was the only material that did not differ significantly from the control sites in granuloma score: granuloma formation due to silk was intermediate between the control score and those for other suture types.

The high body-fluid content of Aplysia, along with their limited ability to osmoregulate,^12,25 likely led to the high degree of variability observed in the amount of fluid present within the subcutis of the controls. Aplysia naturally have a large amount of fluid within the extracellular matrix of their subcutis, which likely led to the observed variability. This high variance within all groups made it difficult to draw conclusions about differences between suture types in respect to this factor. Due to this innate variability, we consider the significant effect between individual animal and edema to be a factor of animal variability and not surgeon variability.

Granuloma formation is a typical reaction to foreign matter such as suture material. Why silk failed to incite the degree of tissue reactivity seen in other species is unclear. One potential cause of Aplysia’s lower reaction to this multifilament suture might be similarity between the structural motifs of silk and proteins of the Aplysia, thus dampening the innate immune response of Aplysia to the presence of silk when compared with the response mounted by vertebrate immune systems. However, even though both Aplysia and Bombyx (silkworms) are invertebrates, there is considerable phylogenetic divergence between them.

A second potential cause of the better-than-expected performance of silk is linked to the lack of observable bacteria on histopathologic review of the samples. The chemical nature and coating of a suture, along with the type of bacteria (gram-negative versus gram-positive), are important contributing factors to bacterial attachment.⁴ Decreased adherence of bacteria to the multifilament sutures such as silk and polyglactin 910 would lead to a reduction in overall tissue reaction. Perhaps innate antimicrobial peptides produced by Aplysia helped to prevent or slow bacteria from being wicked into the suture tract and establishing an infection.²⁹ However, similar peptides are present in several taxa that show marked tissue reaction to silk, including frogs,^10,21,22 fish,^7,19,22 and mammals.^2,15

Monofilament sutures are less reactive in general than multifilamentous sutures in several species,^1,6,13,28 including 3 aquatic species.^8,11,24 Compared with 4 other suture materials, polyglyconate induced the mildest tissue reaction in the koi carp.¹¹ Monofilament nylon was recommended as the preferred suture material in the African clawed frog.²⁴ A similar study in loggerhead sea turtles (Caretta caretta) found that poliglecaprone 25 and polyglyconate produced the least tissue reaction among the 4 suture materials examined.⁸ These findings are in contrast to the results of the current study, in which monofilament sutures induced as much tissue reaction as did multifilament materials.

Suture loss was greater from biopsy sites than from the study sites. Because most of the simple interrupted sutures were maintained during the study, we attribute loss of sutures to suture pattern and incision tension rather than to the suture material itself. Aplysia are highly mobile animals that contort their bodies in many directions to explore their environment. In the course of these activities, the body wall, parapodia, and foot all expand and contract, leading to increased tension on suture patterns that incorporate more tissue, such as the cruciate pattern. Our observations of the movements of the Aplysia suggest to us that there is more movement in a side-to-side or craniocaudal direction. These movements cause most of the skin's expansion to occur in a craniocaudal orientation, with less expansion dorsoventrally. A simple interrupted pattern oriented vertically, as done for the suture study, might have been more successful for closing skin defects. This type of pattern would accommodate expansion of the space between sutures, allowing movement without placing excessive tension on individual sutures.

Of the 5 suture materials evaluated, none appeared to be statistically superior for use in Aplysia. However, silk induced less granuloma formation than did the other sutures, such that the granuloma score for silk did not differ from that of untreated tissue. A reduction in the degree of granuloma formation is a favorable quality that would make silk a preferred suture in these animals. However, in all cases, all suture materials produced significant tissue reaction as compared with control tissue. Therefore, sutures should not remain in place longer than is necessary to provide adequate wound healing.