To our knowledge, this study is the first in which intramuscular injections of sterile water were investigated scientifically in humans. The main findings were that injections of sterile water into the masseter muscle induced similar pain intensity (peak pain intensity), pain duration, VAS auc, and pain area as hypertonic saline i.e. significantly higher pain intensity, longer duration and larger pain spread compared to isotonic saline. These findings suggest that sterile water can be used as an alternative acute experimental pain model to hypertonic saline. However, in similarity to hypertonic saline, sterile water did not affect the PPT, why it does not fully mimic clinical masseter myalgia.
Intramuscular injections of sterile water caused a short-lasting pain of a moderate intensity. This is in line with other commonly used experimental pain models, such as hypertonic saline or glutamate injections into the masseter muscle that cause pain less than 10 min with an average peak-pain intensity around NRS 5–6 (10, 16, 30). However, sterile water had a somewhat different pain profile than hypertonic saline. Peak pain intensity was reached directly after injection and then the pain immediately started to decline but with a slower rate than hypertonic saline. Hypertonic saline, on the other hand, reached its peak pain intensity after about one min after injection. The pain then stayed on a higher level than for sterile water for approximately 1.5 min. There were both similarities and differences between sterile water and hypertonic saline in pain characteristics. For example, both sterile water and hypertonic saline evoked pain of significantly higher intensity and pain area when compared to isotonic saline. Regarding hypertonic saline, this is in accordance with previous studies (11, 31, 32). On the other hand, sterile water induced pain with a 120% longer duration than hypertonic saline (NS), which is an advantage over hypertonic saline. Another advantage is that sterile water is bought ready-made in sterile plastic ampules, while hypertonic saline needs to be diluted to the correct concentration in the clinic/laboratory. This possesses a risk for varying concentrations and the sterility of the solution.
In contrast to the effect on pain variables, our findings showed no significant changes in PPT values after injections of any substance. Also regarding PPT after hypertonic saline injection, previous studies show contradictory results. A few studies report reduced muscle PPT, i.e. mechanical sensitization (11, 33), while other studies report no mechanical sensitization (10, 18, 34–37). These findings indicate that experimentally induced muscle pain with hypertonic saline do not necessarily lead to muscle sensitization. Based on the findings from this single study it seems that sterile water shows the same lack of mechanical sensitization. This is a disadvantage for an experimental model for clinical myalgia. Nevertheless, these results together show that sterile water may be used as an alternative experimental pain model to hypertonic saline (10).
The pain mechanism for hypertonic saline is not clear but is theorized that pain is caused by a shift in sodium concentrations leading to depolarization of excitable membranes, which in turn cause free muscle afferents to be activated by a higher osmolality (19, 38). In a previous study, sodium concentrations were recorded to be 40.5% higher after injections of hypertonic saline (39). Hypertonic saline has an osmolarity of 2002 mOsm/l which is higher than that of plasma (308 mOsm/l) (28). According to other studies, solutions with different osmolarity than blood irritate biological tissues and thus, may cause pain (24, 25). Similar, a shift in the osmolarity may explain the pain evoked by sterile water. However, sterile water is a hypotonic solution with an osmolarity lower than plasma (28). When a hypotonic solution is injected in biological tissues pain will occur due to a lower osmolarity as well as a lower osmotic pressure (40). After injection of a hypotonic saline solution, water from the extracellular fluid flows into the cell causing it to swell. This influx of water is due to a change in ion concentrations as well as a change in osmotic pressure (41). To restore the osmotic gradient balance, sodium and potassium channels are opened and an outflow of sodium and potassium will occur. The ions will then affect the muscle afferents to induce pain. When the osmotic imbalance is restored the cell will return to its normal size (42).
There were some sex differences regarding pain characteristics with a longer pain duration in men for all substances and larger pain area in women after experimentally induced pain by hypertonic saline. However, there were no differences between sexes in peak pain intensity or VAS auc. Earlier studies report inconsistent results regarding sex differences in pain characteristic after algesic injections. For example, in one study women had a longer pain duration and larger pain area than men after experimentally induced muscle pain with acidic saline (17). Yet other studies report no differences in pain duration (11, 31), but larger pain area in women after hypertonic saline injection (17, 31, 43). Furthermore, although results from this study report no differences in peak pain intensity, other experimental studies show that women rate pain higher than men after glutamate-induced pain in the masseter muscle (16, 44). One possible explanation could be the differences in the particular group of individuals included in this study i.e. the group was somewhat younger compared to previous studies (11, 17, 31), hence with less or no previous pain experiences in life. Since most experimental studies also include quite few participants sample selection probably could explain the somewhat different results across studies.
PPT did not differ significantly between sexes after injections, although PPT decreased by 20% 5 minutes after injection of sterile water in women. Hence, the results are in concordance with a few studies that reported no sex difference in PPT after hypertonic saline injection (11, 34), and glutamate injection (16, 44). However, the PPT values were generally higher in men, which supports results from some previous studies of sex differences in masseter PPT (45, 46). Taken together, our results regarding sex differences support data from previous systematic reviews with meta-analysis that show that women in general rate similar pain stimuli as more painful (47) and are more sensitive to pain than men (48).
In accordance with previous studies, there was no adverse effects by the injected solutions. Hypertonic saline and isotonic saline have previously been used in other studies without any side effects (31). Since sterile water often is used to dilute hypertonic saline, no side effects were anticipated. The pain induced by the injected solutions had vanished after a few minutes and had no long-lasting effects. In animal studies on rabbits and rodents, sterile water showed the less tissue damage than isotonic saline (26)
Some strengths and limitations of this study need to be addressed. One strength was that the experiment used a randomized, double blind and placebo controlled design which is considered to be the “gold standard” of human experimental studies (49). Also, using both a passive and an active control substance verifies the outcome of sterile water. Furthermore, the one-week wash out in-between sessions allowed the muscle tissue to restore in order not to cause any false pain experiences by remaining tissue damage. Another strength is that the injection depth in the masseter muscle was controlled so placement and depth of the needle could not affect the results. A limitation of this study was that the menstrual cycle for women was not considered. However, all women participating in the experiment were in different phases of the menstrual cycle and thus not likely to affect the results.