Histologic changes deemed important in the OVA BALB/c mouse model in previous studies were found in the current effort, but this study identified a set of six histological findings that the second study set showed was 97.4% sensitive and 100% specific for mice underwent an allergic challenge. Acute stress was evaluated by multiple comparisons and never found to be of importance in this model. Alveolar dilatation and hemorrhage, not being shown to be associated with allergic challenge, stress, detectable IL-4, detectable IL-5, epithelial cell concentration, or mitotic activity, likely resulted from bronchoalveolar lavage (BAL). Although the semi-quantitative grading system did not prove successful in the first study group, quantitative measures proved more promising: in the second study group, challenged mice, when compared with control mice, had a small but significant decrease in bronchial epithelial cells per 0.1 mm basement membrane and markedly increased odds of having mitoses in the largest non-tracheal respiratory passage. In the third study, time since exposure was associated with a decline in the proportion of inflamed non-tracheal respiratory passages, a decline that was increased by the presence of mitoses in the two largest non-tracheal respiratory passages. The latter finding allows one to posit as grading parameters the proportion of inflamed respiratory passages and mitotic activity.
Prior studies of the BALB/c OVA mouse asthma model that evaluated pulmonary histological changes in some cases restricted their interest to the trachea or main bronchus [7, 27, 28]. Collins  provided results of histologic evaluations of 15 mice, with photographs that appear to show a complete response for acutely sensitized mice and an incomplete response for chronically sensitized mice. Because immunization is known to in and of itself be responsible in humans and cats for an anaphylaxis, [29, 30] the findings suggest that any study comparing immunized and allergic mice carefully evaluate the histological findings in all animals to exclude those who experienced allergic pulmonary inflammation resulting from the immunization. Four sensitized animals in this study developed a complete histological response. The allergic response, if it occurred, occurred at an earlier stage is suggested by the absence of detectable IL-5 in three of the mice.
When histological findings are obliterated by therapy, the change of a histological response, and not the degree of the response, is documented. Soluble IL-15Rα  and rolipram  obliterate inflammation, extirpating, not simply diminishing, the response. Antigen pulsed dendritic cells alter the histological appearance by eliminating the eosinophils, which does not merely diminish the asthmatic attack, but actually reduces the airway responsiveness to methylcholine beneath that observed in control animals ; the latter paper is of note, as it identified as important interalveolar and subpleural inflammatory infiltrate. Desmet [23, 24] showed diminution to an apparent incomplete response after treatment with nuclear factor- κB (NF-κB) and activator protein-1 decoy oligodeoxyribonucleotides. Photographs in this paper show peri-bronchial inflammation, the reason for the designation, in this paper, of "bronchoarterial space." Inflammation is not uniformly distributed about the airways, but shows a marked predilection for the side adjacent to arteries and arterioles. For this reason, statements such as "peri-bronchial and peri-vascular" [22, 25] confuse unless the latter term references pulmonary veins. However, IL-4 and IL-13 were reported to induce airway responsiveness in the absence of inflammation .
de Siqueira  found wrinkling of the epithelium and dilatation of the alveolar spaces, which they demarcated as emphysematous to be of importance. Because their mice underwent BAL, it became of interest to see if the wrinkling and the alveolar dilatation might be unrelated to an asthmatic challenge. Some degree of bronchial epithelial wrinkling was seen in at least one airway in all mice; because quantifying the degree of wrinkling and the proportion of airways with wrinkling was difficult at best. This study concentrated on two relatively easily observed indications of lung injury not seen in every mouse--alveolar dilatation (denoted by these authors as emphysema) and hemorrhage. The results of the present study showed the latter two findings bore no relationship to stress, allergic challenge, IL-4, IL-5, the number of epithelial cells per 0.1 mm of basement membrane, or mitoses, meaning they likely reflected injury from BAL. Although such remodeling might be seen in mice that had not undergone BAL as a result of an allergic insult, the changes would be irrelevant to the determination of the presence or absence of histologic alterations resembling asthma after BAL; therefore we suggest studies of remodelling after BAL must take into account the potential for BAL-induced changes.
Brewer  included a grading system that evaluated the percentage of bronchioles involved. The promising results may have been in part due to its comparing mice that lacked an allergic/asthmatic response to mice that bore one; among all but the four mice strains who had both extremely low airway reactivity and inflammation, there appeared to be no relationship between the inflammatory scores and reactivity. The present study affirmed the diagnostic utility of the proportion of respiratory passages involved by inflammation. Pastva  devised a semi-quantitative inflammatory grading system that served as the basis of the one used in this study. We did not notice differences in cell height, which in the exercised/stressed group shown by their figure 1b appeared similar to that seen in another published study of an unexercised mouse group . Furthermore, it was unclear what the authors meant by degree of inflammation. Evaluation of the modified grading system showed no results that were inexplicable by chance, but did suggest the direction of the remaining grading studies. Because hematoxylin and eosin is not the best stain to evaluate mucin, any conclusion with respect to mucin is of limited value.
Different mouse strains and different allergic/asthma models yield different histological appearances [8, 10–12]. For each mouse strain, a histological definition of the murine histologic alterations resembling asthma should be derived and verified. The current findings apply only to the BALB/c OVA mouse model. Strain of mouse and antigen/model selection will influence the inflammatory response and persistence of airway responsiveness .
Varying the time between last exposure and sacrifice decreases the acute response with time [6, 31]. Molecular markers may also be of use; Duan [14, 15] evaluated the effectiveness of p38α mitogen-activated protein kinase anti-sense oligonucleotide and U0126, a specific MAPK/ERK kinase inhibitor on inflammation as outcomes to evaluate a scoring system . Concerning grading parameters, although the number of bronchial epithelial cells/mm2 was slightly lower, consistent with the notion that larger cells were replacing smaller cells [32, 33], the small size of the differences precludes their routine use. Image analysis is likewise time consuming, but does have the advantage of some objectivity. One study showed differences in the concentration of eosinophils  over time, as well as, with the use of immunohistochemistry, changes in smooth muscle thickness . When time after stress was used as a means to provide a graded level of mediator, the proportion of respiratory passages showing inflammation and the presence of mitoses proved important, suggesting the possibility of their utility in a histological grading system. The histological score might be a better guide for the severity of allergic pulmonary inflammation than analysis of BAL fluid or chemical analyses of whole tissue sample. Eisenbarth  assessed the importance of differing concentrations of lipopolysacharide on the asthmatic response. Photographs of hematoxylin and eosin stained sections showed no difference with respect to the dosage of lipopolysaccharide, notwithstanding marked changes in the laboratory findings. In the grading criteria proposed here, the proportion of inflamed respiratory passages and mitoses, are of interest because they lie in opposition to one another. One fault of prior grading systems is the assumption that all findings are additive.
The two different stresses deserve further comment. Psychostimulants such as amphetamine and cocaine can induce a withdrawal syndrome minimally obfuscated by somatic signs, being mostly a psychological/neurochemical stressor . Withdrawal from repeated amphetamine stimulation results in distress and depressive-like behaviors in rodent models [34, 35]. In humans, the relatively transient withdrawal" or "distress" syndrome is characterized by depression, including psychomotor alterations, dysphoria, anxiety, anhedonia and anergia . Post stimulants can also induce psychotic states in humans. In rats, repeated administration of psychomotor stimulants, such as amphetamine leads to behavioral sensitization and a progressive augmentation of behavioral responses to drug administrations, which persist even after long withdrawal periods [34, 36]. Although the forced swim test would seem to simply be a acute physical stressor, it is also a model of the mental stress of depression [37–39] during acute stress, being a widely used pharmacologic model for assessing antidepressant activity in the rodent laboratory [35, 39]. Researchers describe a "depressive" behavior as the floating response because the swimming behavior ceases and a state of despair or subjective helplessness is observed [36, 38]. Neither form of stress proved important in any overall way that could not have been explained by chance, supporting the utility of the definition in animals that had undergone stress and animals that had not undergone stress. Because two amphetamine-stressed control animals developed a complete response and had detectable IL-5, relationship of stress to the induction of an acute allergic response must be further explored. Future analyses of allergic asthma might wish to quantify differences in BAL fluid with respect to inflammatory mediators and extensively analyze the effects of changes in immunization protocols to assess differences between mice that are physically stressed and mice that only experience psychological stress.
In another report by our laboratory, an analysis of mean levels of IL-4, but not IL-5, were elevated among mice exposed to the forced swim test compared to control mice Thus, while control and non-allergic-challenged stressed mice did not differ in the proportion of mice with detectable IL-4 in BAL, the mean levels of IL-4 were elevated among stressed mice. Mice with histologic alterations resembling asthma responded to stress differently than did control mice, such that mean IL-4 levels did not differ between stressed and non-stressed allergic animals By contrast, although mean IL-5 levels were elevated among allergic challenged animals, stress actually reduced mean IL-5 levels in BAL among allergic mice
Limitations of this study include the absence of image analysis, special stains, gene expression analyses, and immunohistochemical studies, and lack of data on airway resistance. The definition and proposed grading system is limited to the BALB/c OVA mouse model. Similar studies should be undertaken for other mouse models before identifying a histologic alterations resembling asthma. There exists no reason a priori for levels of any specific inflammatory mediator in any specific mouse model to correlate with the degree of severity of an allergic challenge because inflammatory mediators may induce a response without a proportional histological response. The proposed grading system must be verified by a physiologic grading system that includes measures of air way resistance or pulmonary hyperresponsiveness. Despite these limitations, the value of the histological definition lies in the simplicity and speed with which the most commonly used murine model of allergic pulmonary inflammation can be confirmed. With practice, the identification of a response takes less than one minute and can be performed on animals that have little or no other measures of allergic pulmonary inflammation or asthma.