Early inflammatory response of knee ligaments to prolotherapy in a rat model

Abstract

Prolotherapy is an alternative injection-based therapy for chronic musculoskeletal pain. Three different proliferants, D-glucose (dextrose), phenol-glucose-glycerine (P2G), and sodium morrhuate, used in prolotherapy are hypothesized to strengthen and reorganize chronically injured soft tissue and decrease pain through modulation of the inflammatory process. Our hypothesis is that commonly used prolotherapy solutions will induce inflammation (leukocyte and macrophage infiltration) in medial collateral ligaments (MCLs) compared to needlestick, saline injection, and no-injection controls. MCLs of 84 Sprague- Dawley rats were injected one time at both the tibial and femoral insertions. Immunohistochemistry (IHC) was used to determine the inflammatory response at three locations (tibial and femoral insertions and midsubstance) 6, 24, and 72 h after dextrose injection compared to saline- and no-injection controls and collagenase (positive control) (n = 4). qPCR was used to analyze gene expression 24 h postinjection (n = 4). Sodium morrhuate, P2G, and needlestick control were also investigated after 24 h (n = 4). In general, inflammation (CD43+, ED1+, and ED2+ cells) increased after prolotherapy injection compared to no-injection control but did not increase consistently compared to saline and needlestick control injections. This response varied by both location and proliferant. Inflammation was observed at 6 and 24 h postinjection but was resolved by 72 h compared to no-injection controls (p < 0.05). CD43+ leukocytes and ED2+ macrophages increased compared to needlestick and saline-injection control, respectively, 24 h postinjection (p < 0.05). Prolotherapy injections created an inflammatory response, but this response was variable and overall, not uniformly different from that caused by saline injections or needlestick procedures.

(c) 2008 Orthopaedic Research Society.

Figures

Figure 1. Study Design

Rats were injected at time zero and euthanized at six, 24, and 72 hours post-injection. Rats euthanized at all time points were injected with the most commonly used PrT solution (dextrose) or saline (as a control) or collagenase (as a positive control). The same no-injection control rats were used at the six and 24 hour time points. Additional PrT solutions (sodium morrhuate and P2G) and control (needlestick) were investigated 24 hours post-injection. MCLs at all time points were analyzed using immunohistochemistry (IHC) for inflammation, and MCLs at 24 hours post-injection were analyzed using quantitative polymerase chain reaction (qPCR) for inflammatory genes.

Figure 2. Immunohistochemistry Images

No-injection controls (left column: A, C, E) and dextrose-injected ligaments six hours post-injection (right column: B, D, F). Dextrose-injected ligaments had similar numbers of CD43-positive lymphocytes (arrow) (B) compared to no injection (A). Dextrose-injected ligaments had more ED1-positive macrophages (arrows) (D) compared to no injection (C). Dextrose-injected ligaments had more ED2-positive macrophages (arrows) (F) compared to no injection (E).

Figure 3. Normalized Number of CD43+ Leukocytes 24 hours Post-Injection at the Tibial Insertion, Midsubstance, and Femoral Insertion

At the tibial insertion, there was a significant increase in the number of CD43+ leukocytes with saline, sodium morrhuate, P2G, and collagenase (positive control) injection compared to no injection (*p

Figure 4. Normalized Number of ED1+ Macrophages six hours Post-Injection at the Tibial Insertion, Midsubstance, and Femoral Insertion

Six hours post-injection at the tibial insertion, there was an increase in the number of ED1+ macrophages with saline and collagenase (positive control) injection compared to no injection (* p

Figure 5. Normalized Number of ED2+ Macrophages Six hours Post-Injection at the Tibial Insertion, Midsubstance, and Femoral Insertion

Six hours post-injection, there was an increase in the number of ED2+ macrophages in saline- and dextrose-injected ligaments compared to no injection at the tibial and femoral insertions (*p

Figure 6. Normalized Number of ED2+ Macrophages 24 hours Post-Injection at the Tibial Insertion, Midsubstance, and Femoral Insertion

Twenty-four hours post-injection at the femoral insertion, there was an increase in the number of ED2+ macrophages with needlestick, dextrose, sodium morrhuate, and collagenase injection compared to no injection (* p