Therapeutic effect of C-type natriuretic peptide on persistent pain in a rat knee arthritis model

Ethics

This study was approved by the Institutional Animal Care and Use Committee (IACUC) of this university (approval number A2022-104A). This study was reported in accordance with ARRIVE guidelines.

Materials

CNP, which comprises of the C-terminal 22 amino acid of human origin, was purchased from Peptide Institute Inc. (Tokyo, Japan). MIA and paraformaldehyde (PFA) were purchased from Sigma-Aldrich (St Louis, MO, USA). Ethylenediaminetetraacetic acid (EDTA), Fast Green FCF, and isoflurane were purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). Safranin O was purchased from Chroma-Gesellschaft-Schmidt and Co (Stuttgart, Germany). Anti-calcitonin gene-related peptide (CGRP) antibody was purchased from Peninsula Laboratories LLC (San Carlos, CA, USA). Mayer’s hematoxylin solution, 1% eosin alcohol solution, and Masson stain solution B (Ponceau xylidine, acid fuchsin, and azophloxine solutions) were purchased from Muto Pure Chemicals Co, Ltd (Tokyo, Japan).

MIA-Induced rat arthritis model

Twenty male Wistar rats (Charles River, Tokyo, Japan) at 9−11 weeks of age and 310−350 g in weight were used. Rats were randomly divided into two groups, with 10 rats in each group; one group received CNP, whereas another group received a vehicle solution (phosphate-buffered saline [PBS]). Isoflurane was used to anesthetize rats (2% in oxygen, flow rate of 2 L/min). Under anesthesia, an intra-articular injection of MIA (1 mg in 30 μL PBS) was injected into the right knee joint, as described previously. ⁹ CNP and vehicle group animals were injected with CNP (1 µg in 20 μL of 5% glucose solution) and PBS (20 μL) into the right knee joint, respectively, for six consecutive days from eighth day after MIA administration. The rats were kept under a 12/12-h light/dark cycle with food and water ad libitum. On day 20, the rats were euthanized using carbon dioxide, and the right knee joints were dissected for histological and immunohistochemical evaluations. To confirm fibrosis, the knee joints of six rats were harvested on the eighth day independently and analyzed (Supplemental Figure S1).

Evaluation of pain avoidance behavior

Weight-bearing imbalance was evaluated at day 0 (pre-injection), and on days 1, 4, 7, 14, 18, and 19 after MIA injection using an incapacitance tester (Linton Instrumentation, Norfolk, UK) (Figure 1).^10,11,13 The rats were acclimatized to the tester three times a week before MIA injection. The rats were placed in a clear plexiglass case for the first 5 min until they settled down with their left and right limbs on an independent force plate. Measurements were performed 100 times per measurement, and the percentage weight of the right hind limb (MIA side) was calculated. The percentage weight distribution of the ipsilateral hind limb was calculated as follows: ¹⁶ OPEN IN VIEWER

[Weight distribution of ipsilateral hind limb rate (%) = ipsilateral weight/(ipsilateral weight + contralateral weight) × 100].

Tactile hypersensitivity of the hind paw was measured on the same day as the incapacitance test using von Frey Hairs (Touch Test Sensory Evaluator, North Coast Medical Inc. Morgan Hill, CA, USA) with weights ranging from 1 to 26 g. Rats were placed in a wire mesh cage for over 10 min until they settled down. The hair was applied to the central plantar surface of the hind paw from below the cage in ascending order starting with 1 g hair. Hair of a certain weight was pressed until it bent and held for 5 s. If the hind paw was lifted off the floor of the cage, this was considered a withdrawal response. A test was performed five times at 5 s intervals per session. If a withdrawal response occurred more than three times in a session, a one-rank lighter weight was recorded as the withdrawal threshold. If it did not, the next increased hair weight was applied. After determining the withdrawal threshold of the left side, the right side was tested in the same manner. The protocol was modified from that of a previous study.^17,18 The pain avoidance behavior tests were conducted in a standardized manner without blinding after training with multiple people beforehand.

Histological and immunohistochemical examinations

The dissected knee joints were fixed in 4% paraformaldehyde at pH 7.4 for 1 week, demineralized in 20% EDTA in PBS (pH 7.4) for 3 weeks, and embedded in paraffin. Sagittal sections were cut to a thickness of 5 µm. The sections were de-paraffinized and stained with hematoxylin/eosin (H/E), Masson trichrome, or Safranin-O/fast green. The IFP inflammation score was used to semi-quantitatively evaluate the severity of synovial inflammation and fibrosis (Supplemental Table S1). Cartilage degeneration was evaluated using OA Research Society International (OARSI) score as previously reported.^9,19 IFP fibrosis and inflammatory cell infiltration were evaluated in the mid sagittal section where the anterior cruciate ligament was visible. The medial cartilage was evaluated after a spacing of 200 µm (Figure 1).

Sections were deparaffinized in xylene, rehydrated in graded alcohol, and soaked in PBS for immunohistochemical staining of CGRP-positive nerve fibers. Endogenous peroxidases were quenched in methanol for 15 min using 0.3% hydrogen peroxidase. For optimal antigen retrieval, the sections were rinsed three times with PBS, fixed again in 4% PFA for 15 min, pretreated with Target Retrieval Solution (Dako, Glostrup, Denmark) in citrate buffer for 30 min at 98°C, and incubated for 20 min at room temperature. To avoid non-specific antibody binding, the sections were rinsed with PBS and blocked with 5% normal goat serum for 30 min at room temperature (Vector Laboratories, Burlingame, CA, USA). Sections were subsequently incubated with rabbit anti-CGRP polyclonal antibody (1:250 dilution in PBS containing 1% bovine serum albumin [BSA]; Peninsula Laboratories LLC, San Carlos, CA) in a humid chamber overnight at 4°C. As a negative control, normal rabbit serum was used as a primary antibody instead of anti-CGRP antibody (Supplemental Fig S2). Sections were rinsed with PBS and incubated for 30 min with biotinylated goat anti-rabbit IgG secondary antibody (Vector Laboratories). Vectastain ABC reagent (Vector Laboratories) was used to visualize the CGRP-positive nerve fibers. Hematoxylin was used to counterstain sections. The number of CGRP‐positive fibers was counted as previously described.^10,11,13 Histochemical and immunohistochemical evaluations were performed by three independent raters in a blinded manner, and the Intraclass Correlation Coefficient (ICC) (2,1) were 0.77–0.93.^20,21 The ICC values were classified as follows: values ≤0.5 indicated poor reliability, 0.5–0.75 indicated moderate reliability, 0.75–0.90 indicated good reliability, and 0.90 ≤ indicated excellent reliability. The ICC values of H/E stain for cell infiltration, Masson trichrome stain for IFP fibrosis, CGRP stain for CGRP-positive nerve fiber, and Safranin O/Fast Green Stain for cartilage degeneration were 0.86, 0.77, 0.86, and 0.93, respectively.

Intra-Articular injection of CNP after IFP fibrosis improves pain avoidance behavior in an MIA-Induced rat arthritis model

After extensive practice, a self-evaluation conducted prior to this experiment revealed an almost perfect success rate for intra-articular injection. Moreover, in the histological examination conducted after the animals were sacrificed, increased synovial cellularity was observed in all cases. Based on this finding, we believe that the success rate of intra-articular injection was close to 100%. There were no injection complications. Histological evaluations indicated extensive synovial hyperplasia with fibrotic lesions in the IFP on day 8 after MIA injection (Supplemental Figure S1), and CNP was administered on day 8 in this study. To evaluate time-course changes in pain avoidance behavior, an incapacitance test was conducted according to the time course indicated in Figure 1. A decrease in the percentage of weight-bearing on the ipsilateral limb was observed in both the CNP and vehicle groups by day 4 after MIA injection (Figure 2(a)). No clear difference was observed between the groups on day 14. However, the CNP group showed a mild increase in weight-bearing in the ipsilateral hind limb than that of the vehicle group on day 18 (Figure 2(a)). On day 19, a significant improvement was observed in weight-bearing in the CNP group (Figure 2(a)). The Von Frey hair test, which indicates the mechanical hypersensitivity of the hind paw, was also conducted on the same time course as that of the incapacitance test. The withdrawal threshold of the right hind paw in both groups gradually decreased by day 8 after the MIA injection. CNP did not increase the paw withdrawal threshold by day 19 (Figure 2(b)).OPEN IN VIEWER

Intra-Articular administration of CNP during the inflammation subsiding period reduced the severity of IFP fibrosis

To verify whether the increase in weight-bearing after CNP administration during the inflammation subsiding period correlates with the suppression of IFP degeneration, inflammation, fibrosis, and invasion of CGRP-positive sensory nerve fiber endings were examined at day 20 after MIA administration. H/E staining indicated that in the vehicle group, almost all of the fat cells had already been replaced by fibroblastic cells, with an increase in cell density in the IFP region at day 20 (Figure 3(a), vehicle). In contrast, CNP administration during the inflammation subsiding period significantly suppressed the replacement of fat cells by fibroblastic cells, with a decrease in cell density in the IFP region (Figure 3(a), CNP). Histological grading analysis supported these results (Figure 3(b), cellularity score; vehicle vs. CNP; p = 0.03). Deposition of collagen fibers in IFP, which is indicated by blue staining during the Masson trichrome staining, was also reduced after CNP administration during the inflammation subsiding period (Figure 3(c)). Semi-quantitative IFP fibrosis grading analysis indicated that although the difference was not statistically significant, CNP administration reduced collagen fiber deposition in the IFP region (Figure 3(d), vehicle vs. CNP; p = 0.08). Immunohistochemical evaluation indicated that the number of CGRP-positive nerve fiber endings in the CNP group was significantly lower than that in the vehicle group (Figure 4, vehicle vs. CNP; p = 0.04).OPEN IN VIEWER

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Figure 4.

CGRP-positive nerve fiber invasion was decreased by CNP administration (a) Arrowheads indicate CGRP-positive nerve fiber endings. The CNP group shows fewer CGRP-positive nerve fiber endings than that of the vehicle group. (b) Numbers per unit area of CGRP-positive nerve fiber endings in IFP regions are counted. Dots represent each sample. Asterisk indicates p < 0.05 by Student t-test (PBS n = 10; CNP n = 9; Decreased n numbers in the CNP group due to a technical issue).

Articular cartilage degeneration was not inhibited by intra-articular injection of CNP during the inflammation subsiding period

In our previous study, we showed that CNP administration during the acute inflammatory stage significantly inhibited articular cartilage degeneration on day 28. ¹³ To verify whether CNP administration during the inflammation subsiding period also has anti-catabolic effects on articular cartilage, histological evaluation using Safranin-O/fast green was performed. As shown in Figure 5, extensive loss of proteoglycan in the loading surface of the articular cartilage was observed in both groups (Figure 5(a)). OARSI grading was consistent with these results, as no significant difference was observed between the two groups (vehicle vs. CNP: 95%CI -6.85−2.20; p = 0.29) (Figure 5(b)).OPEN IN VIEWER