This data confirms the critical role of neutrophil recruitment in the early inflammatory phase of wound healing in older rats. WP was found to recover the wound closure rate in the older rats to a similar level to that of the young rats. These observations are in accordance with those of a previous study that suggested that the Kombucha fungus accelerates the healing quality
Wound healing entails three distinct phases: an initial inflammatory phase, a proliferation phase and, finally, the production and reorganization of the extracellular matrix, leading to tissue repair or regeneration
[26–28]. Defects in the inflammatory phase of healing directly cause a failure in the subsequent processes of fibroblast growth and collagen synthesis
[29, 30]. The inflammatory phase recruits leukocytes that produce growth factors and remove debris from the wound
. Impairment of leukocyte recruitment is associated with delayed wound healing
[31–33]. Neutrophils release highly active antimicrobial substances, proteinases
 and inflammatory cytokines which also have crucial roles in the healing of wounds. The precise role of neutrophils in wound healing is still a rich subject of scientific controversy since little is known about exactly how neutrophils affect wound healing. Here, we have investigated neutrophil recruitment during wound healing using an in vivo rat model, together with the effects of dietary WP supplementation.
Recent work has suggested that depletion of neutrophils accelerates the healing process
. In contrast, in this study, we found that wounds in young rats, which showed normal healing, were infiltrated by greater numbers of neutrophils, especially in the inflammatory stage, followed by a gradual depletion in the next stages. On the other hand, our results point to a marked deficiency in the number of neutrophils in the wounds of aged rats, with this being accompanied by impairment of the healing process. This result is in accordance with Nishio et al.,
 who also argued that the depletion in neutrophils served to impair the rate of healing of wounds in older mice. In addition, Brubaker et al.,
 have argued that reduced neutrophil chemotaxis and infiltration contributes to delayed resolution of cutaneous wound infection with advanced age.
The inflammatory phase of healing can be divided into an early phase, with neutrophil recruitment, and a late phase with the appearance and transformation of monocytes
. Normally, neutrophils are recruited to the site of the skin injury and are present for 2–5 days unless the wound gets infected
. Some studies, however, have shown that, in some cases, such as with wounds in diabetics, a delayed contribution of neutrophils at the wound site subsequent to the inflammatory stage suppresses the healing process due to the accompanying high inflammation and oxidative stress
[35, 37, 38]. Here we found that neutrophil infiltration of the older dermis delayed to the eighth day post wounding. At this late stage, it is more likely that neutrophils may produce persistently high levels of free radicals and inflammatory cytokines, as suggested by Fulop et al.
 who mentioned that aging causes multiple defects in PMN function, notably increased production of ROS with high concentrations of inflammatory cytokines induced by a high level of free radicals via NFkB. This may explain the prolonged process of wound closure in rats from the older group: in other words, prolonged wounds remain in a chronic inflammatory state
 which leads to abnormal wound repair
. On the other hand, WP-lactoferrin has an ability to stimulate immune responses involving neutrophils and macrophage cytotoxicity
. This further explains the early infiltration of neutrophils in old rats supplemented with WP. Moreover, it is concluded that lactoferrin acts as an anti-inflammatory by regulating the levels of TNF-α and IL-6
. This explains the significant decrease in TNF-α in the serum of old rats supplemented with WP. In addition, increasing the level of the powerful antioxidant, glutathione, by WP supplementation of the older rats (Figure
1D), may have induced oxidative stability, scavenging the free radicals after the inflammatory phase of wound healing. This should suppress the induction of inflammatory cytokines and may encourage the next stages of normal healing.
The alteration of neutrophil functions observed here might be caused by alterations in the signal transduction pathways
. Changes in membrane fluidity affect PMN functions, such as chemotaxis and superoxide anion production
. Furthermore, neutrophils are necessary for producing MMPs, which further degrade the damaged tissue and produce chemokines, which in turn attract additional neutrophils
. Here, WP was found to significantly up-regulate the MIP-1α and CINC-1 mRNA expression in old rats supplemented with WP at 2 days after wounding with respect to old rats, in which MIP-1α and CINC-1 mRNA expression was delayed to the 8th day after wounding. This data is a further evidence on the early and the delayed infiltration of neutrophils in old rats supplemented with WP and old rats, respectively.
Neutrophils are uniquely sensitive to a vast array of chemoattractants including a multitude of chemokines (CINC-1, MIP-1a, MIP-1b, MIP-2, KC, and others). Chemoattractants signals choreograph the transit of leukocytes out of the mainstream of blood and into tissues at sites of inflammation.
The re-epithelialization process is underpinned by keratinocytes at the wound edges and by epithelial stem cells from hair follicles or sweat glands
[43, 44]. In the present study, wound margin epithelia of older rats supplemented with WP were increased in both their size and the extent of their migration at the edge of the wound, resulting in a moderate closing of the wound by the fourth day after wounding. The release of EGF, TGF-a, and FGF is thought to stimulate epithelial cell migration and proliferation, with keratinocytes then migrating over the provisional extracellular matrix. Once wound closure is achieved, keratinocytes undergo stratification and differentiation to restore the barrier
. Our results demonstrated that WP may encourage these successive events of re-epethialization, resulting in a complete covering of the wound region by the eighth day after wounding.
At the beginning of re-epithelialization from the wound edges, neovascularization is activated
[46–48]. Neutrophils release mediators such as TNF-α, IL-1ß and IL-6, which serve to stimulate VEGF and IL-8 for an adequate repair response
. Binding of VEGF to their receptors on the endothelial cells of existing vessels activates the intracellular signalling cascades
[36, 49] and cause the endothelial cells to proliferate and migrate into the wound, a process known as 'sprouting’
. In our study, the number of blood vessels and the extent of collagen deposition in the wounded region were significantly reduced in the older rats in comparison to the younger ones, but WP was found to significantly restore these indicators in the older group. Since WP also served to restore the number of neutrophils a positive correlation between the number of neutrophils and the formation of new vessels is proved.
Taken together, our results confirm that neutrophils are more likely to play a central role in the early inflammatory phase of wound healing in the elderly. Depletion of neutrophils in this stage may affect: 1) the secretion of inflammatory cytokines; 2) the production of chemokines; 3) the proliferation and migration of keratinocytes; 4) the initiation of angiogenesis, and 5) the formation and deposition of collagen fibres. Hence, this study has shown that WP may exert a beneficial effect on wound healing in older rats by restoring normal levels of neutrophil infiltration. This data may provide critical insights into future nutritional intervention strategies designed to enhance wound healing in the elderly.