Spider housing and silk collection
Trichonephila edulis spiders were housed as previously described.24 The animals live free within an own room with temperatures between 20°C (winter) and up to 30°C (summer) and a relative humidity in a range from 40 to 60%. Animals were watered daily with tab water and fed twice a week with crickets (Acheta domesticus). Under laboratory conditions T. edulis mates all over the year. Due to the free keeping in the room, targeted mating is not necessary. After fertilization, the female spider leaves her web and builds a cocoon (egg sac), which can contain several hundred eggs. Shortly after finishing the cocoon, the animal returns to its web and cocoons can be collected. The eggs, which are packed in a bundle and coated with particles, were removed (Fig. 1).25
Remaining cocoon silk was macroscopically cleaned from visible particles. According to the established standard cleaning procedure the cocoon silk was washed thoroughly three times in aqua distillate by vortexing for 30 s each, then sterilized by autoclaving and dried.
Animal experiments
All animal experimental protocols were approved by the Lower Saxony State Office for Consumer Protection and Food Safety (LAVES; Approval No 33.8-42502-04-21/3771). The ARRIVE guidelines were followed while reporting. All methods were performed in accordance with relevant guidelines and regulations. Six adult male rats (LEW/HanZtm, Central animal facility, Hannover Medical School, Germany) with a body weight of 410.8 g +/- 8.3 g were implemented in the study. The rats were housed in pairs (cage type IVs, Uno Roestvaststaal BV, The Netherlands) at room temperature between 20 and 24°C, relative humidity of 45–65% and 14/10 h day–night cycle with free access to standard diet (1324 TPF, Altromin, Lage/Westphalia, Germany) and water. Animals were handled several times a week for five weeks to adapt them to the personnel and surrounding.
Anesthesia and analgesia
Surgery was performed under isoflurane anesthesia (2–5 vol% in Oxygen; Isofluran CP, cp-pharma, Burgdorf, Germany) and additional local anesthesia at the tail base (10 mg/kg Lidocainhydrochlorid 2%, s.c., bela-pharm, Vechta, Germany). The rats received carprofen (5 mg/kg s.c., Rimadyl®, Zoetis, Berlin, Germany) 30 minutes and buprenorphine (0.05 mg/kg s.c., Bupresol®, vet, cp-Pharma, Burgdorf, Germany) 15–20 minutes before the operation as pre-emptive pain medications and an additional subcutaneous postoperative buprenorphine injection approximately 4 hours after surgery.
The administration of carprofen was continued for five days postoperatively. Carprofen could have been given for extended periods of pain as further treatment option.
Enrofloxacin (5 mg/kg, s.c., Baytril®, Bayer, Leverkusen, Germany) was applied preoperatively and four days after surgery as antibiotic prophylaxis.
Surgical procedure
The hairy tail base as well as the remaining rat tail were shaved and disinfected with povidone-iodine solution. To reduce possible bleeding during surgery, an elastic band was placed at the tail base. Afterwards the rat was placed in prone position on a warm pad. The tail was covered with sterile drapes and a second careful disinfection was performed. The skin incision was made dorsally at the level coccyx (Co) 4–5 to Co 5–6, approximately 3 cm in length. The vertebral level concerned was identified by palpation. The soft tissue was gently prepared down to the disk area. To prepare a disk defect, an 18 G cannula was stabbed in the intervertebral space. The defect was implemented in two neighboring discs. One defect was left empty (blank sample), in the other one a small pellet of spider silk was filled. If necessary, bleeding was stopped by compression. Wound closure was performed in single stitch technique with a resorbable suture (Vicryl 4 − 0, Ethicon) (Fig. 2).
Stress evaluation
The animals were observed for six weeks overall after surgery to evaluate the stress level of the surgery and to control the postoperative regime. Therefore, a daily check-up was accomplished for the first two weeks. Depending on the tension level further controls were performed daily or reduced to three times a week. The overall burden corresponds to the highest level in a sub-category. The stress level and subsequent measuring were recorded on a rat-specific score sheet adapted to the test execution. They are based on the directive 2010/63/EU of the European Parliament and Working Group of Berlin Animal Officers. The official authorization was obtained by LAVES.
Table 1
Score sheet for the postoperative evaluation of general condition, behavior and surgical wound
score value | body weight | general condition | behavior | surgical wound |
1 | • weight gain • steady • weight loss ≤ 9.9% | • coat smooth, shiny, orifices clean • defecation and urine secretion normal • eyes bright | • awake, attentive, curious • physiological body posture and gait | • no secretion • no swelling • no hyperthermia • no redness • no dehiscence • tail movement normal |
2 | • weight loss 10-14.9% | • coat matted focally • coat defect • defecation and urine secretion normal | • calm, attentive, allotriophagy (pica-behavior) • reduced movement • reduced cleaning behavior • species specific body posture | • possible secretion • possible swelling • possible hyperthermia • possible redness • possible dehiscence • tail movement possibly reduced |
3 | • weight loss 15-19.9% | • coat matted, shaggy • neglected orifices, slightly affected | • very calm • inattentive • weak • sternoabdominal posture • reduced motion | • possible secretion • possible swelling • hyperthermia < 24 h • possible redness • possible dehiscence • tail movement possibly reduced |
4 | • weight loss ≥ 20% | • dirty coat • sticky or moist orifices • defecation and urine secretion impaired • atypical body posture • eyes sticky and sunken | • apathetic, lateral posture • pronounced hyperkinetic • stereotypes of behavior • coordination disorders • automutilation | • possible secretion with pus • possible swelling • hyperthermia < 24 h • possible redness • dehiscence • tail movement reduced |
Table 2
Score sheet for stress evaluation and assigned measures
Score value | Assessment | Measures |
1 | no stress | • none |
2 | minor stress | • careful observation (at least once a day) • adjuvant therapy (infrared heat lamp, pain medication, soaked food) |
3 | moderate stress | • careful observation (at least once a day) • careful observation (twice a day) on the occurrence of social disorder/decreased general health • adjuvant therapy (infusion, pain medication and/or antibiotic treatment) • symptoms > 48 h: euthanasia |
4 | intense stress | • euthanasia |
The behavior of each rat was observed for at least 5 minutes. To evaluate the movement of the tail properly, the rat was placed on a table to run free. In addition, the determination of mechanical pain was performed by Von Frey-filaments.26, 27 The Testing was implemented two days preoperatively and on day two, seven, 14, 21, 28 and 44 after surgery. Therefore, the rat was placed on a lattice, acclimatized for 5 min and then stimulated with Von Frey-filaments (Marstock Nerve test, Schriesheim, Germany) with strength of 2 g (19.6 mN) down to 0.5 g (4.9 mN) on the ventral tail for six seconds each. The filament had to bend slightly. With each filament strength, five repetitions were done.
If the rat avoided the filament, shook off the base of the tail or licked itself within six seconds after the filament stimulus, the response was evaluated as “positive”. If there was none of the reactions mentioned, the response was evaluated as “missing”.
The Von Frey-filament test was continued in descendant filament strength until no more “positive” reactions could be provoked. Then, the testing was performed in an ascendant order.
Histological preparation
Six weeks after surgery animals were finally anesthetized and sacrificed. Vertebrae were cut with a diamond saw (EXAKT Advanced Technologies GmbH, Norderstedt, Germany) and the subsequent areas (Co 4–5 and Co 5–6) were prepared. The adjacent areas Co 3–4 and Co 6–7 were taken as healthy controls.
Samples were fixed in 4% buffered formalin (Medite Medical GmbH, Burgdorf, Germany) for at least two days at 4°C and embedded in methyl methacrylate (Technovit® 9100 New, Heraeus-Kulzer, Hanau, Germany) for histological analyses according to the manufacturer’s instructions and established protocols.28
4 µm thin longitudinal sections were cut with a microtome (RM 2155, Leica, Bensheim, Germany) and associated tungsten carbide knives (Leica, Bensheim, Germany), placed on poly-L-lysine coated glass slides, stretched with 90% ethanol, covered with a polyethylene foil, pressed and dried for at least 48 h at 37°C. For Toluidine blue staining (0.1%, Sigma-Aldrich Chemie, Taufkirchen, Germany, 30 sec), sections were deacrylated in xylene (2 × 10 min) and 2-methoxyethylacetate (1 × 10 min) and rehydrated with a decreasing alcohol series. Afterwards, slices were washed with distilled water, dehydrated in an increasing alcohol series, and mounted with Eukitt® (Labonord, Mönchengladbach, Germany). After drying at room temperature for at least 24 h light microscopic examination was performed with an Axioskop 40 microscope supplemented by a polarisation optics to countercheck for silk detection. Images were taken with an AxioCam Mrc digital camera and Axio Vision Software (all Carl Zeiss, Oberkochen Germany).
Evaluation was performed descriptively.