Hypochlorous Acid Solution Is Safe for Intracavitary Lavage: Examination in a Rodent Model

General procedure details

Animals were anesthetized via intraperitoneal injection of the ketamine/xylazine/acepromazine mixture: 40 mg/kg, 8 mg/kg, and 4 mg/kg, respectively.⁶ After ensuring adequate anesthesia, the animal was transferred to the operative field and placed on an oxygen nose cone. Clippers were used to remove hair from the surgical field. Procedure-specific details are described in the following sections. All 3 procedures ended with skin closure using interrupted nonabsorbable sutures. Following the procedure, 0.05 mg/kg of buprenorphine was administered for analgesia and animals were closely monitored until recovery.

Laparotomy

A ventral midline incision was made to expose the peritoneum. The peritoneum was lifted and incised with scissors to enter the abdominal cavity. The incision was then extended to expose all intra-abdominal organs (Fig 1A). The abdominal cavity was then irrigated with 5 mL of the selected solution, which was left to instill for 2 minutes. The solution was then carefully aspirated from dependent areas of the abdomen with a syringe and an 18-gauge catheter. Suctioning near organs was avoided to limit iatrogenic injury. Irrigation and aspiration were then repeated with an additional 5 mL of solution. Before the final interrupted skin closure, the peritoneum was closed with a running nonabsorbable suture.

Thoracotomy

Under adequate general anesthesia, animals were intubated with a 16-gauge catheter as an endotracheal (ET) tube over a blunted spinal needle that was attached to a small animal ventilator (RoVent Jr, Kent Scientific Corporation, Torrington, CT). Placement was confirmed by observation of chest rise. For the entirety of the procedure, animals were ventilated on room air with weight-based tidal volumes. A 1- to 2-cm incision was made in the skin of the right posterior-lateral chest at approximately the right fourth and fifth ribs. The intercostal space was then incised above the fifth rib to expose the thoracic cavity. A self-retaining microsurgical retractor was introduced between the ribs and opened slowly until direct visualization of the lung expanding was obtained (Fig 1B).

Through this opening, the thoracic cavity was irrigated with 3 mL of the selected solution using a 16-gauge catheter on a 5-mL syringe. After instilling for 2 minutes, easily accessible fluid was carefully aspirated while avoiding direct suction on lung tissue. This process was repeated with an additional 3 mL of solution. After the second lavage, the syringe was disconnected from the catheter that remained in the thoracic cavity. The muscle surrounding the thoracotomy was then reapproximated, and a purse string suture was placed around the catheter. An empty 5-mL syringe was then placed on the catheter. To mitigate postoperative pneumothorax, gentle aspiration was applied to the syringe as the catheter was removed while cinching the purse string. The ET tube was removed after the animal demonstrated normal respiration while disconnected from the ventilator.

Laminectomy

With the animal in the prone position, a 2- to 4-cm incision was made over the L5-L6 spinous processes. The paraspinous muscles were then dissected free, and a small rongeur was used to remove the most accessible spinal process. Ligamentum flavum was then removed to expose the spinal cord (Fig 1C). Using a micropipette, 50 µL of the selected solution was applied and allowed to instill for 5 seconds. Any remaining accessible fluid was then aspirated off again with the pipette. The supraspinal ligament and the surrounding muscle were then reapproximated with a nonabsorbable running suture before the interrupted skin closure.

Euthanasia and necropsy

Following the procedures, animals were monitored for surgical site infections and general systemic changes. On postoperative day 5, the animals were euthanized under inhaled isoflurane anesthesia. Animals that did not survive until day 5 were necropsied at the time of death. Surgical sites were opened, and procedure-specific organs were collected and formalin-fixed as described in Table 1.

Histology

Hematoxylin and eosin (H&E) staining was performed on formalin-fixed organs. In addition, apoptosis was measured with immunohistochemistry (IHC) using a primary anti-caspase-3 antibody and a secondary fluorescent antibody. Images were analyzed using ZEN software from ZEISS Microscopy (Carl Zeiss Microscopy, LLC, White Plains, NY).

Morbidity and mortality

All animals survived until postoperative day 5 except for 2 of the 3 rats in the NaOCl thoracotomy group. One of these animals developed an increased work of breathing approximately 2 hours after the ET tube was removed and was unable to recover, while the other was unable to breathe independently off the ventilator at the end of the procedure. There was no appreciable pneumothorax in either animal on necropsy, but the lungs of both animals were impressively inflamed compared with lungs of NS- and HOCl-treated animals.

There were no differences in pain or behavior between the NS and HOCl groups as determined by grimace scores and observations of behavior and eating habits. For all 3 procedures, animals treated with NS or HOCl appeared to return to baseline health and activity within 6 hours after surgery. All animals treated with NaOCl, however, demonstrated increased pain and lethargy and decreased appetite that required treatment with additional fluid resuscitation and analgesia.

On necropsy, none of the animals were found to have developed a surgical site infection regardless of the irrigant used (Fig 2). Organs of NS- and HOCl-treated animals were grossly similar, while inflammation marked by erythema and fibrosis was noted in the lungs of all 3 NaOCl thoracotomy animals (Fig 3) and intra-abdominal organs of 2 of 3 NaOCl laparotomy animals.

Figure 2. Representative images of surgical scars of (a) laparotomy, (b) thoracotomy, and (c) laminectomy on day 5.

Figure 3. Gross specimens of lungs treated with (a) HOCl solution and (b) NaOCl solution. NaOCl-treated lungs demonstrated increased fibrosis and hemorrhage compared with NS and HOCl. HOCl indicates hypochlorous acid; NaOCl, sodium hypochlorite or Dakin's solution; NS, normal saline.

Histology

There were no differences on H&E staining between animals treated with NS and HOCl. Structural integrity of all tissues examined in these groups was maintained without evidence of inflammation or fibrosis. In contrast, significant inflammation, fibrosis, and hemorrhage were seen on the surfaces of tissues exposed to NaOCl lavage (Fig 4).

Figure 4. Representative H&E images of spleen (top row) and lung (bottom row) capsules exposed to the 3 different irrigants. Increased fibrosis and underlying hemorrhage was seen in the NaOCl-treated tissues (*). H&E indicates hematoxylin and eosin; NS, normal saline; HOCl, hypochlorous acid; NaOCl, sodium hypochlorite or Dakin's solution.

Similar findings were found on IHC regarding the presence of apoptotic cells. The fluorescent secondary antibody identified caspase-3 in the cytoplasm of cells undergoing apoptosis. The surfaces of NaOCl-treated tissues, however, consistently demonstrated an increased presence of apoptosis compared with NS and HOCl. Apoptotic cells were especially prominent in the small bowel mesentery and lungs of NaOCl-treated animals (Fig 5).

Figure 5. Representative images of IHC from (B) bowel wall and (M) mesentery. No apoptotic cells were seen in the NS or HOCl groups. These cells were noticeably present in the mesentery of NaOCl-treated animals (circled). IHC indicates immunohistochemistry; NS, normal saline; HOCl, hypochlorous acid; NaOCl, sodium hypochlorite or Dakin's solution.