Intramuscular Electroporation With the Pro-Opiomelancortin Gene in Rat Adjuvant Arthritis

I-Chuan Chuang; Chien-Ming Jhao; Chih-Hsun Yang; Hsien-Chang Chang; Chien-Wen Wang; Cheng-Yuan Lu; Yao-Jen Chang; Sheng-Han Lin; Pao-Lin Huang; Lin-Cheng Yang

Disclosures

Arthritis Res Ther. 2004;6(1) 

In This Article

Results

Two groups of rats were used in the study: normal rats and rats treated with CFA to induce arthritis.

Rats in group A (normal rats) were assigned to five subgroups (SG), I, II, III, IV and V, as follows: SG I (n = 7), intramuscular electroporation with 200 µg of pCMV–POMC; SG II (n = 7), intramuscular electroporation with 20 µg of pCMV–POMC; SG III (n = 7), intramuscular electroporation with PBS; SG IV (n = 7), intramuscular injection with 200 µg of pCMV–POMC without electroporation; and SG V (n = 7), injection of PBS without electroporation, as control group.

The serum levels of ACTH and endorphin in SG III and SG IV were not significantly increased in comparison with the SG V control group. SG II rats, which received 20 µg of pCMV–POMC by electroporation, showed a modest increase in the levels of ACTH and endorphin, but this was not statistically significant when compared with SG V. In contrast, SG I, which received intramuscular electroporation with 200 µg of pCMV–POMC, showed increases in the serum level of ACTH and endorphin that peaked at 2586 and 1859 pg/ml at week 1, respectively. This gradually decreased to 750 and 351 pg/ml at week 3, respectively. At all time points of the reported experiment, the serum level of endorphin in SG I was much higher than that of SG IV (from 12-fold at 7 days to 3.5-fold at 21 days). The time courses of serum ACTH and endorphin after intramuscular electroporation with the gene encoding POMC are shown in Figs 1 and 2.

We performed CFA injection at 1 week after muscle-targeted transfer of 200 µg of pCMV–POMC or PBS using intramuscular electroporation. CFA-treated rats were divided into five subgroups (SG), VI, VII, VIII, IX and X, as follows: SG VI (n = 7), intramuscular electroporation with 200 µg of pCMV–POMC followed by injection with CFA; SG VII (n = 7), intramuscular electroporation with PBS followed by injection with CFA; SG VIII (n = 7), intramuscular PBS injection with CFA only (this group was used to determine whether CFA itself significantly induced the production of endogenous ACTH and endorphin); SG IX (n = 7), treated as SG VI except that, 30 minutes before the nociceptive test at 7 days after CFA injection, naloxone (1 mg/kg) was administered intraperitoneally to determine whether any analgesic effect was mediated through the opioid receptors; and SG X (n = 7), intramuscular electroporation with 200 µg of pCMV-Script vector alone added to the negative control to demonstrate that pCMV-Script vector alone could not induce POMC expression.

The serum levels of ACTH and endorphin in SG VII were not significantly increased in comparison with the SG V control. Moreover, there was no difference between SG VII and the other control group (SG VIII, PBS and CFA only). We can therefore answer that electroporation itself did not induce significant expression of ACTH or systemic effects. The serum levels of ACTH and endorphin in SG VII were not significantly increased in comparison with the SG V control. In contrast, SG VI, which received intramuscular electroporation with 200 µg of pCMV–POMC, showed increases in the serum level of ACTH and endorphin that peaked at 2904 and 1642 pg/ml, respectively, at week 1. This gradually decreased to 870 and 716 pg/ml, respectively, at week 3. The time courses of serum of ACTH and endorphin concentrations after intramuscular electroporation with the gene encoding POMC in CFA-treated rats are shown in Figs 1 and 2.

Time course of POMC gene injection and electroporation on the blood levels of ACTH. Results are means ± SEM. Statistical comparisons between groups were made by analysis of variance; individual comparisons were made with the post-hoc test. *P < 0.05.

Time course of POMC gene injection and electroporation on the blood levels of beta-endorphin. Results are means ± SEM. Statistical comparisons between groups were made by analysis of variance; individual comparisons were made with the post-hoc test. *P < 0.05.

The thermal nociceptive thresholds of control rats (SG I–IV) including injection with 200 µg of pCMV–POMC and electroporation did not show any difference from the PBS (SG V) injection group (P > 0.05) (data not shown).

The thermal hyperalgesia of CFA rats (SG VI) injected with 200 µg of pCMV–POMC and electroporation was significantly improved at 3, 5 and 7 days after CFA injection in comparison with the groups receiving PBS with electroporation (SG VII) or only CFA injection (SG VIII) (P < 0.05; Fig. 3). It is noteworthy that pCMV–POMC plus naloxone (SG IX) showed similar thermal thresholds to SG VII, SG VIII and SG X (pCMV vector alone) (P > 0.05;Fig. 3).

Thermal hypersensitivity responses during CFA injection. The POMC and electroporation group (SG VI) suppressed the CFA-induced pain (*P < 0.05), showing a significant difference between rats receiving electroporation and CFA (SG VII), CFA only (SG VIII) and electroporation of pCMV vector alone (SG X), whereas naloxone reversed the analgesic effects of POMC (SG IX). Results are means ± SEM.

To determine the anti-inflammatory effects caused by intramuscular electroporation gene therapy with POMC, we measured paw swelling in CFA rats in subgroups SG VII, which received intramuscular electroporation with PBS, and SG VI, which received 200 µg of pCMV–POMC. SG VIII rats, which received only CFA injection, were used as a control group. The peak effect was noted at day 7 in SG VI (Fig. 4). Rats injected with PBS with electroporation showed a similar degree of swelling to that of the control group (CFA only). However, naloxone administered at 7 days after CFA injection was without any significant effect in paw swelling in polyarthritic rats that received 200 µg of pCMV–POMC (SG IX).

Paw swelling during CFA injection in control rats (SG VIII) and rats receiving POMC gene and electroporation (SG VI) and electroporation and paw CFA injection (SG VII). POMC gene injection and electroporation (SG VI) suppressed the swelling. Results are means ± SEM. Statistical comparisons between groups were made by analysis of variance; individual comparisons were made with the post-hoc test. *P < 0.05.

A high level of transfer of the gene encoding POMC was obtained after electroporation of muscle tissue. This was determined by analysis of the muscle endorphin level by RIA (Fig. 5). The peak effect (650 pg/ml) was noted at day 5 in SG VI. This gradually decreased to 189 pg/ml at day 14 after CFA injection.

Effect of POMC injection on levels of endorphin in muscle at days 3, 5, 7 and 14 after injection with CFA. Results are means ± SEM. Statistical comparisons between groups were made by analysis of variance; individual comparisons were made with the post-hoc test. *P < 0.05, showing a significant difference between rats receiving electroporation (SG VII) and CFA only (SG VIII).

Muscle endorphin mRNA was detected by RT–PCR from week 1 to week 3 (Fig. 6). RT–PCR products of the expected size (450 bp) were obtained with RNA extracted from six pieces of muscle tissue transfected with pCMV–POMC. This was true in all the POMC-transfected animals. Control tissues failed to show any pCMV–POMC mRNA by RT–PCR. These findings indicate that pCMV–POMC mRNA was transcribed in the transfected muscle (Fig. 6).

RNA levels of β-endorphin were measured at weeks 1, 2 and 3 after injection with PBS (SG III) or 200 µg of POMC cDNA (SG I) and electroporation. Results are means ± SEM. Statistical comparisons between groups were made by two-tailed unpaired t-test. *P < 0.05, showing a significant difference between the control groups and the POMC gene and electroporation group.

Intramuscular injection of 200 µg of pCMV–POMC followed by electroporation was performed to see the effects of transfection in muscle tissue and the location of endorphin. Peak fluorescence was noted at week 1 in the electroporated muscle (Fig. 7). Muscle injected with DNA without electroporation showed weaker immunoreactivity than that in the control group (PBS injection without electroporation).

Confocal micrographs of POMC gene electroporation on endorphin expression in muscle. (A) Immunohistochemical detection of endorphin immunoreactivity in the negative control that omitted the primary antibody. A muscle nucleus is labelled by 4,6-diamidino-2-phenylindole (blue). (B) Overexpression of endorphin immunoreactivity (green) in the POMC-treated group. Scale bar, 40 µm. (C) High-magnification image (from (B)) of endorphin-positive puncta shows the detailed distribution of endorphin in a muscle cell. Scale bar, 20 µm. (D) Endorphin-positive puncta are absent from muscle electroporated with pCMV-Script vector alone. Scale bar, 20 µm. (E, F) No significant inflammatory cell infiltration or muscle damage on haematoxylin/eosin staining of POMC-electroporated muscle (E) and untreated muscle (F). Original magnification, ×200. Scale bar, 100 µm.

We cannot exclude the possibility of low and transient tissue damage induced by electroporation. However, haematoxylin/eosin staining did not demonstrate abnormal inflammatory cell infiltration or necrosis at the voltage used.

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