2.4.2. LT protocol
Leeches were placed on the rats using forceps 5 mm away
from the wound (Fig. 3). The leeching time was counted
from the time the anterior sucker of the leech attached to
the site near the wound. Regular therapeutic application
times are 30e90 minutes for humans during LT. However,
because of anatomical and physiological differences between
humans and rats, the aforementioned regular therapeutic
application time is fatal for rats (personal
observation). After 2 minutes, the leeches were detached
by applying salt at the back of the leeches. Detachment of
leeches by applying salt might induce vomiting, which
might lead to infection. However, because leeches were
not satiated with blood and their feeding time was short,
neither vomiting nor consequent infection was observed. At
the end of the procedure, the leeches were deposited in
90% ethanol.
2.5. Wound appearance analyses
Each wound region was digitally photographed at the indicated
time intervals, and the respective wound areas were
calculated by Photoshop, version 7.0 [12]. Changes in the
wound area were expressed as the percentage of the initial
wound area [37].
Figure 1 A medicinal leech (Hirudo orientalis) used in the
study (scale Z cm).
Figure 2 The location of full-thickness incisional wound at
the back of rat near the median line (scale Z cm).
Figure