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Longitudinal evaluation of immunohistochemical findings of milk aspiration: An experimental study using a murine model
Forensic Science International 209 (2011) 183–185
Contents lists available at ScienceDirect
Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Longitudinal evaluation of immunohistochemical findings of milk aspiration: An experimental study using a murine model Tomonori Nagai, Miwako Aoyagi, Eriko Ochiai, Kentaro Sakai, Kyoko Maruyama-Maebashi, Kenji Fukui, Kimiharu Iwadate * Department of Forensic Medicine, The Jikei University School of Medicine, Nishishinbashi 3-25-8, Minato-ku, Tokyo 105-8461, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 August 2010 Received in revised form 19 November 2010 Accepted 20 January 2011 Available online 17 February 2011
To examine the longitudinal change of pathological findings of the lung and other organs in milk aspiration, an experimental study using a murine model was carried out. Either 0.5 or 1.0 ml cow’s milk was instilled into the trachea of rats. From immediately after to 14 days after instillation, the animals were sacrificed, and the lungs, liver, kidneys, and spleen were removed. The results of immunostaining with anti-human a lactalbumin antibody indicated that not only the lung but also the kidney and spleen showed a positive reaction against the antibody over time. Experimentally aspirated milk was detectable in alveoli until 2 days after instillation. It was also detectable in renal tubules from 1 to 6 h after instillation. Macrophages containing granules of aspirated milk were observed in splenic red pulp from 3 h to 14 days after instillation. Detection of aspirated milk in other organs except the lung would be clear evidence of intravital milk aspiration and would suggest previous or recurrent milk aspiration. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: Sudden infant death Milk aspiration Immunostaining
1. Introduction Sudden infant death is one of the most important challenges in forensic medicine. While almost all infants experience some degree of gastroesophageal reflux (GER) [1] and mild milk aspiration is not rare in infant death cases [2], incidental massive milk aspiration may cause asphyxia due to airway obstruction. The relationship between milk aspiration and sudden infant death syndrome (SIDS) has been discussed in some reports [3,4]. The postmortem diagnosis of milk aspiration remains difficult, as Moran [5] initially outlined in 1953. Immunohistochemistry using antibodies against milk components is useful for identifying milk in lung sections [6,7]; however, its applicability to the pathological diagnosis of intravital milk aspiration has not been sufficiently validated. Although it is difficult to resolve this problem by clinical studies, few experimental reports have focused on the morphological diagnosis of milk aspiration. In our previous animal study, we reported that in pathological findings of lung sections obtained within an hour after experimental milk aspiration, it is difficult to distinguish significant intravital aspiration from insignificant aspiration during the agonal state [8]. However, further questions remain regarding the time until aspirated milk is immunohistochemically detectable in the lungs and whether it can be detectable in other organs. It is
* Corresponding author. Tel.: +81 3435 7305; fax: +81 3 3435 7194. E-mail address: [email protected] (K. Iwadate). 0379-0738/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2011.01.026
said that GER and recurrent aspiration of gastric contents may induce many subacute or chronic pulmonary diseases, such as aspiration pneumonia [9,10]. If it is possible to identify previous or recurrent milk aspiration by pathological findings in autopsy cases, it will be helpful for the diagnosis of these subacute or chronic diseases. On the other hand, for example, in cases of fresh water drowning, aspirated water is absorbed into the capillaries of alveoli and circulates to the other organs. Detecting aquatic plankton by diatoms from the liver, kidney and other organs except the lung is important for the diagnosis of drowning [11]. In a similar way, aspirated milk may circulate to the other organs, except the lung. Detection will be clear evidence of intravital milk aspiration. To elucidate these questions, we performed an experimental study using a murine model. The aim of the present study was to examine longitudinal changes of pathological findings of the lung and other organs in experimental cases of milk aspiration. 2. Materials and methods Our experiments were carried out on 10-week-old Wistar rats (Sankyo, Japan). All procedures were approved by the Animal Care and Use Committee of Jikei University School of Medicine. Forty rats were divided into two groups. In 20 rats, 0.5 ml cow’s milk (MEIJI, Japan) was instilled intratracheally, and in the remaining 20 rats, 1.0 ml cow’s milk was instilled intratracheally. Intratracheal instillation of milk was performed generally as described by Costa et al. [12]. In this procedure, a fully anesthetized animal rests supine against a restraining stand angled at 60–70 from the horizontal and is held in position. A small incision is made in the neck, and then 0.5 or 1.0 ml cow’s milk is instilled into the trachea. In both groups, 2 rats were sacrificed immediately after, 1, 3, 6, 12 h, and 1, 2, 4, 7, and 14 days after instillation and organs were removed.
[()TD$FIG]
T. Nagai et al. / Forensic Science International 209 (2011) 183–185
184
Fig. 1. Results of immunostaining with anti-human a lactalbumin antibody of the lungs of rats. 0.5 ml milk was injected into the trachea, and the lungs were removed 6 h (left), 1 day (middle), and 2 days later (right).
[()TD$FIG] In both groups, lung, liver, kidney and spleen specimens were fixed in 10% formaldehyde solution and embedded in paraffin. Formaldehyde solution was not injected into the bronchus for fixation. Sections 2–3 mm thick were prepared. Immunostaining with anti-human a lactalbumin antibody (Dako, Denmark), which is thought to have cross-reactivity against cow a lactalbumin, was performed using the Histfine rat kit (Nichirei, Japan). Immunoreactivity against the antibody was examined by light microscopy. Tissue specimens containing at least minimal substance showing a positive reaction to the antibody were judged as positive.
3. Results The results of immunostaining, the volume of instilled milk, and the time interval from the instillation of milk to removal of the organs are listed in Table 1. Fig. 1 shows longitudinal changes of the immunohistochemical findings of the lungs of examined rats. Aspirated milk in alveoli was identified immunohistochemically until 2 days after instillation. Although alveolar macrophages containing vesicles of aspirated milk were seen from immediately after instillation, they were limited throughout the whole period. Neutrophilic infiltration was not seen in any animals. No milk remained in alveoli 4 days after instillation or later. In both groups, a positive reaction against anti-human a lactalbumin antibody was observed on the internal side of renal tubules from 1 to 3 h after instillation. Both groups showed the same staining pattern, foamy or granular (Fig. 2), as reported in previous human studies [6,7]. The amount of instilled milk had little effect on the pathological findings. Macrophages containing vesicles showing a positive reaction against the antibody appeared in splenic red pulp 3 h after instillation and persisted until 14 days after instillation regardless of the volume of instilled milk (Fig. 3); however, when the amount of instilled milk was small (0.5 ml), the reactivity tended to become weak in spleen sections obtained 7 and 14 days after instillation. A positive reaction against anti-human a lactalbumin antibody was not seen in any liver sections in both groups.
Fig. 2. Results of immunostaining with anti-human a lactalbumin antibody of a rat kidney. 1.0 ml milk was injected into the trachea, and the kidney was removed 3 h later.
4. Discussion In general, it is thought that intravitally aspirated milk can easily reach peripheral alveoli by respiratory airflow and be absorbed by capillaries and venulae, and then scavenged from alveoli. In this study, instilled milk could be identified in alveoli until 2 days after instillation, suggesting that immunohistochemical demonstration of aspirated milk itself might be difficult since several days have passed after milk aspiration. In an experimental study using mice by Elidemir et al. [13], milk-laden macrophages were detected in bronchoalveolar lavage fluid by immunostaining 2 h after milk injection. Bjanowski et al. [14] have also reported that latex particles injected into trachea are phagocytized by alveolar macrophages within several minutes of injection. In the
Table 1 Immunostaining results. The results of immunostaining, amount of instilled milk, and time interval from the instillation of milk to removal of the organs are shown. Amount of milk
0.5 ml
1.0 ml
Time intervals
Lung Kidney Spleen Lung Kidney Spleen
0h
1h
3h
6h
12 h
1d
2d
++
++ ++
++
++
++
++
++
++ ++
++ + ++ ++ ++ ++
+ ++
+ ++
++ ++
++
++
++
+: one of two rats showed positive staining; ++: both rats showed positive staining; h: hours; d: days..
4d
7d
14 d
++ ++
++
++
++
++
++
++
++
[()TD$FIG]
T. Nagai et al. / Forensic Science International 209 (2011) 183–185
185
Fig. 3. Results of immunostaining with anti-human a lactalbumin antibody of a rat spleen. 1.0 ml milk was injected into the trachea, and the spleen was removed 3 h later (left) and 14 days later (right).
present study, macrophages containing aspirated milk were observed immediately after instillation; however, their number was so few that their diagnostic value seemed low. Although neutrophilic infiltration was not seen in any animals, if not only milk but gastric juice is aspirated in lungs in clinical human cases, it may induce more a rapid and severe neutrophilic reaction. Of course, the lung are the most important organ to diagnose and evaluate the severity of milk aspiration; however, these results indicate that to diagnose milk aspiration occurring more than several days previously, the applicability of lung immunostaining may be limited. In this study, renal tubules showed a positive reaction to antihuman a lactalbumin antibody from 1 to 3 h after instillation. Macrophages showing a positive reaction were observed in the spleen from 3 h to 14 days after instillation. As in drowning cases, milk components absorbed into pulmonary capillaries might circulate to the kidneys, spleen and other organs. In the kidneys, aspirated milk might be filtrated at the glomerulus and re-taken up into renal tubules. In the spleen, circulating milk components might be phagocytized by macrophages; however, in the liver, Kuppfer cells showed no positive reaction during the period from instillation, regardless of the volume of instilled milk. Even in the lungs, the number of macrophages containing positive vesicles is few, and only the spleen showed a number of macrophages containing positive vesicles. The results of this study are not sufficient to resolve this question and further study is needed. More importantly, although the results of animal studies cannot be directly applied to human cases, the detection of aspirated milk in organs other than the lungs is clear evidence of intravital milk aspiration. Immunohistochemical examination of the spleen using antibodies against milk components may be useful for the pathological diagnosis of previous or recurrent milk aspiration.
References [1] A.C. Hillemeier, Gastroesophageal reflux; diagnostic and therapeutic approaches, Pediatr. Clin. North Am. 43 (1996) 197–212. [2] K. Iwadate, M. Doy, Y. Ito, Screening of milk aspiration in 105 infant death cases by immunostaining with anti-humana lactalbumin antibody, Forensic Sci. Int. 122 (2001) 95–100. [3] B.T. Thach, Sudden infant death syndrome: can gastroesophageal reflux cause sudden infant death? Am. J. Med. 108 (2000) 144S–148S. [4] N. Alex, J.M.D. Thompson, D.M.O. Becroft, E.A. Mitchell, Pulmonary aspiration of gastric contents and the sudden infant death syndrome, J. Paediatr. Child Health 41 (2005) 428–431. [5] T.J. Moran, Milk-aspiration pneumonia in human and animal subjects, Arch. Pathol. Lab. Med. 55 (1953) 286–301. [6] K. Iwadate, N. Sakamoto, S.H. Park, M. Doy, H. Iwase, M. Nagao, T. Takatori, Immunohistochemical detection of human milk components aspirated in lungs of an infant, Forensic Sci. Int. 90 (1997) 77–84. [7] K. Iwadate, M. Doy, Y. Nishimaki, L. Fang, T. Takatori, H. Hasekura, Immunohistochemical examination of the lungs in infant death cases using antibodies against milk components, Forensic Sci. Int. 110 (2000) 19–28. [8] K. Iwadate, M. Aoyagi, K. Sakai, E. Ochiai, S. Abe, K. Maebashi, M. Nakamura, K. Fukui, Feasibility of immunohistochemical examination of lungs to distinguish intravital milk-aspiration from postmortem ‘‘aspiration’’: a experimental study using a murine model, Res. Pract. Forensic Med. 51 (2008) 233–237. [9] A.R. Euler, W.J. Byrne, M.E. Ament, E.W. Fonkalsrud, C.T. Strobel, S.C. Siegel, R.M. Katz, G.S. Rachelefsky, Recurrent pulmonary disease in children; a complication of gastroesophageal reflux, Pediatrics 63 (1979) 47–51. [10] P. Chen, M. Chang, S. Hsu, Gastroesophageal reflux in children with chronic recurrent bronchopulmonary infection, J. Pediatr. Gastroenterol. Nutr. 13 (1991) 16–22. [11] P. Saukko, B. Knight, Diatoms and the diagnosis of drowning, in: Forensic Pathology, third ed., Hodder Arnold, London, 2004pp.406–408. [12] D.L. Costa, J.R. Lehmann, W.M. Harold, R.T. Drew, Transoral tracheal intubation of rodents using a fiberoptic laryngoscope, Lab. Anim. Sci. 36 (1986) 256–261. [13] O. Elidemir, L.L. Fan, G.N. Colasurdo, A novel diagnostic method for pulmonary aspiration in a murine model: immunocytochemical staining of milk proteins in alveolar macrophage, Am. J. Respir. Crit. Care Med. 161 (2000) 622–626. [14] T. Bjanowski, B. Brinkmann, A.M. Stefanec, R.H. Barckhaus, G. Fechner, Detection and analysis of tracers in experimental drowning, Int. J. Legal Med. 111 (1998) 57–61.
Contents lists available at ScienceDirect
Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Longitudinal evaluation of immunohistochemical findings of milk aspiration: An experimental study using a murine model Tomonori Nagai, Miwako Aoyagi, Eriko Ochiai, Kentaro Sakai, Kyoko Maruyama-Maebashi, Kenji Fukui, Kimiharu Iwadate * Department of Forensic Medicine, The Jikei University School of Medicine, Nishishinbashi 3-25-8, Minato-ku, Tokyo 105-8461, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 August 2010 Received in revised form 19 November 2010 Accepted 20 January 2011 Available online 17 February 2011
To examine the longitudinal change of pathological findings of the lung and other organs in milk aspiration, an experimental study using a murine model was carried out. Either 0.5 or 1.0 ml cow’s milk was instilled into the trachea of rats. From immediately after to 14 days after instillation, the animals were sacrificed, and the lungs, liver, kidneys, and spleen were removed. The results of immunostaining with anti-human a lactalbumin antibody indicated that not only the lung but also the kidney and spleen showed a positive reaction against the antibody over time. Experimentally aspirated milk was detectable in alveoli until 2 days after instillation. It was also detectable in renal tubules from 1 to 6 h after instillation. Macrophages containing granules of aspirated milk were observed in splenic red pulp from 3 h to 14 days after instillation. Detection of aspirated milk in other organs except the lung would be clear evidence of intravital milk aspiration and would suggest previous or recurrent milk aspiration. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: Sudden infant death Milk aspiration Immunostaining
1. Introduction Sudden infant death is one of the most important challenges in forensic medicine. While almost all infants experience some degree of gastroesophageal reflux (GER) [1] and mild milk aspiration is not rare in infant death cases [2], incidental massive milk aspiration may cause asphyxia due to airway obstruction. The relationship between milk aspiration and sudden infant death syndrome (SIDS) has been discussed in some reports [3,4]. The postmortem diagnosis of milk aspiration remains difficult, as Moran [5] initially outlined in 1953. Immunohistochemistry using antibodies against milk components is useful for identifying milk in lung sections [6,7]; however, its applicability to the pathological diagnosis of intravital milk aspiration has not been sufficiently validated. Although it is difficult to resolve this problem by clinical studies, few experimental reports have focused on the morphological diagnosis of milk aspiration. In our previous animal study, we reported that in pathological findings of lung sections obtained within an hour after experimental milk aspiration, it is difficult to distinguish significant intravital aspiration from insignificant aspiration during the agonal state [8]. However, further questions remain regarding the time until aspirated milk is immunohistochemically detectable in the lungs and whether it can be detectable in other organs. It is
* Corresponding author. Tel.: +81 3435 7305; fax: +81 3 3435 7194. E-mail address: [email protected] (K. Iwadate). 0379-0738/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2011.01.026
said that GER and recurrent aspiration of gastric contents may induce many subacute or chronic pulmonary diseases, such as aspiration pneumonia [9,10]. If it is possible to identify previous or recurrent milk aspiration by pathological findings in autopsy cases, it will be helpful for the diagnosis of these subacute or chronic diseases. On the other hand, for example, in cases of fresh water drowning, aspirated water is absorbed into the capillaries of alveoli and circulates to the other organs. Detecting aquatic plankton by diatoms from the liver, kidney and other organs except the lung is important for the diagnosis of drowning [11]. In a similar way, aspirated milk may circulate to the other organs, except the lung. Detection will be clear evidence of intravital milk aspiration. To elucidate these questions, we performed an experimental study using a murine model. The aim of the present study was to examine longitudinal changes of pathological findings of the lung and other organs in experimental cases of milk aspiration. 2. Materials and methods Our experiments were carried out on 10-week-old Wistar rats (Sankyo, Japan). All procedures were approved by the Animal Care and Use Committee of Jikei University School of Medicine. Forty rats were divided into two groups. In 20 rats, 0.5 ml cow’s milk (MEIJI, Japan) was instilled intratracheally, and in the remaining 20 rats, 1.0 ml cow’s milk was instilled intratracheally. Intratracheal instillation of milk was performed generally as described by Costa et al. [12]. In this procedure, a fully anesthetized animal rests supine against a restraining stand angled at 60–70 from the horizontal and is held in position. A small incision is made in the neck, and then 0.5 or 1.0 ml cow’s milk is instilled into the trachea. In both groups, 2 rats were sacrificed immediately after, 1, 3, 6, 12 h, and 1, 2, 4, 7, and 14 days after instillation and organs were removed.
[()TD$FIG]
T. Nagai et al. / Forensic Science International 209 (2011) 183–185
184
Fig. 1. Results of immunostaining with anti-human a lactalbumin antibody of the lungs of rats. 0.5 ml milk was injected into the trachea, and the lungs were removed 6 h (left), 1 day (middle), and 2 days later (right).
[()TD$FIG] In both groups, lung, liver, kidney and spleen specimens were fixed in 10% formaldehyde solution and embedded in paraffin. Formaldehyde solution was not injected into the bronchus for fixation. Sections 2–3 mm thick were prepared. Immunostaining with anti-human a lactalbumin antibody (Dako, Denmark), which is thought to have cross-reactivity against cow a lactalbumin, was performed using the Histfine rat kit (Nichirei, Japan). Immunoreactivity against the antibody was examined by light microscopy. Tissue specimens containing at least minimal substance showing a positive reaction to the antibody were judged as positive.
3. Results The results of immunostaining, the volume of instilled milk, and the time interval from the instillation of milk to removal of the organs are listed in Table 1. Fig. 1 shows longitudinal changes of the immunohistochemical findings of the lungs of examined rats. Aspirated milk in alveoli was identified immunohistochemically until 2 days after instillation. Although alveolar macrophages containing vesicles of aspirated milk were seen from immediately after instillation, they were limited throughout the whole period. Neutrophilic infiltration was not seen in any animals. No milk remained in alveoli 4 days after instillation or later. In both groups, a positive reaction against anti-human a lactalbumin antibody was observed on the internal side of renal tubules from 1 to 3 h after instillation. Both groups showed the same staining pattern, foamy or granular (Fig. 2), as reported in previous human studies [6,7]. The amount of instilled milk had little effect on the pathological findings. Macrophages containing vesicles showing a positive reaction against the antibody appeared in splenic red pulp 3 h after instillation and persisted until 14 days after instillation regardless of the volume of instilled milk (Fig. 3); however, when the amount of instilled milk was small (0.5 ml), the reactivity tended to become weak in spleen sections obtained 7 and 14 days after instillation. A positive reaction against anti-human a lactalbumin antibody was not seen in any liver sections in both groups.
Fig. 2. Results of immunostaining with anti-human a lactalbumin antibody of a rat kidney. 1.0 ml milk was injected into the trachea, and the kidney was removed 3 h later.
4. Discussion In general, it is thought that intravitally aspirated milk can easily reach peripheral alveoli by respiratory airflow and be absorbed by capillaries and venulae, and then scavenged from alveoli. In this study, instilled milk could be identified in alveoli until 2 days after instillation, suggesting that immunohistochemical demonstration of aspirated milk itself might be difficult since several days have passed after milk aspiration. In an experimental study using mice by Elidemir et al. [13], milk-laden macrophages were detected in bronchoalveolar lavage fluid by immunostaining 2 h after milk injection. Bjanowski et al. [14] have also reported that latex particles injected into trachea are phagocytized by alveolar macrophages within several minutes of injection. In the
Table 1 Immunostaining results. The results of immunostaining, amount of instilled milk, and time interval from the instillation of milk to removal of the organs are shown. Amount of milk
0.5 ml
1.0 ml
Time intervals
Lung Kidney Spleen Lung Kidney Spleen
0h
1h
3h
6h
12 h
1d
2d
++
++ ++
++
++
++
++
++
++ ++
++ + ++ ++ ++ ++
+ ++
+ ++
++ ++
++
++
++
+: one of two rats showed positive staining; ++: both rats showed positive staining; h: hours; d: days..
4d
7d
14 d
++ ++
++
++
++
++
++
++
++
[()TD$FIG]
T. Nagai et al. / Forensic Science International 209 (2011) 183–185
185
Fig. 3. Results of immunostaining with anti-human a lactalbumin antibody of a rat spleen. 1.0 ml milk was injected into the trachea, and the spleen was removed 3 h later (left) and 14 days later (right).
present study, macrophages containing aspirated milk were observed immediately after instillation; however, their number was so few that their diagnostic value seemed low. Although neutrophilic infiltration was not seen in any animals, if not only milk but gastric juice is aspirated in lungs in clinical human cases, it may induce more a rapid and severe neutrophilic reaction. Of course, the lung are the most important organ to diagnose and evaluate the severity of milk aspiration; however, these results indicate that to diagnose milk aspiration occurring more than several days previously, the applicability of lung immunostaining may be limited. In this study, renal tubules showed a positive reaction to antihuman a lactalbumin antibody from 1 to 3 h after instillation. Macrophages showing a positive reaction were observed in the spleen from 3 h to 14 days after instillation. As in drowning cases, milk components absorbed into pulmonary capillaries might circulate to the kidneys, spleen and other organs. In the kidneys, aspirated milk might be filtrated at the glomerulus and re-taken up into renal tubules. In the spleen, circulating milk components might be phagocytized by macrophages; however, in the liver, Kuppfer cells showed no positive reaction during the period from instillation, regardless of the volume of instilled milk. Even in the lungs, the number of macrophages containing positive vesicles is few, and only the spleen showed a number of macrophages containing positive vesicles. The results of this study are not sufficient to resolve this question and further study is needed. More importantly, although the results of animal studies cannot be directly applied to human cases, the detection of aspirated milk in organs other than the lungs is clear evidence of intravital milk aspiration. Immunohistochemical examination of the spleen using antibodies against milk components may be useful for the pathological diagnosis of previous or recurrent milk aspiration.
References [1] A.C. Hillemeier, Gastroesophageal reflux; diagnostic and therapeutic approaches, Pediatr. Clin. North Am. 43 (1996) 197–212. [2] K. Iwadate, M. Doy, Y. Ito, Screening of milk aspiration in 105 infant death cases by immunostaining with anti-humana lactalbumin antibody, Forensic Sci. Int. 122 (2001) 95–100. [3] B.T. Thach, Sudden infant death syndrome: can gastroesophageal reflux cause sudden infant death? Am. J. Med. 108 (2000) 144S–148S. [4] N. Alex, J.M.D. Thompson, D.M.O. Becroft, E.A. Mitchell, Pulmonary aspiration of gastric contents and the sudden infant death syndrome, J. Paediatr. Child Health 41 (2005) 428–431. [5] T.J. Moran, Milk-aspiration pneumonia in human and animal subjects, Arch. Pathol. Lab. Med. 55 (1953) 286–301. [6] K. Iwadate, N. Sakamoto, S.H. Park, M. Doy, H. Iwase, M. Nagao, T. Takatori, Immunohistochemical detection of human milk components aspirated in lungs of an infant, Forensic Sci. Int. 90 (1997) 77–84. [7] K. Iwadate, M. Doy, Y. Nishimaki, L. Fang, T. Takatori, H. Hasekura, Immunohistochemical examination of the lungs in infant death cases using antibodies against milk components, Forensic Sci. Int. 110 (2000) 19–28. [8] K. Iwadate, M. Aoyagi, K. Sakai, E. Ochiai, S. Abe, K. Maebashi, M. Nakamura, K. Fukui, Feasibility of immunohistochemical examination of lungs to distinguish intravital milk-aspiration from postmortem ‘‘aspiration’’: a experimental study using a murine model, Res. Pract. Forensic Med. 51 (2008) 233–237. [9] A.R. Euler, W.J. Byrne, M.E. Ament, E.W. Fonkalsrud, C.T. Strobel, S.C. Siegel, R.M. Katz, G.S. Rachelefsky, Recurrent pulmonary disease in children; a complication of gastroesophageal reflux, Pediatrics 63 (1979) 47–51. [10] P. Chen, M. Chang, S. Hsu, Gastroesophageal reflux in children with chronic recurrent bronchopulmonary infection, J. Pediatr. Gastroenterol. Nutr. 13 (1991) 16–22. [11] P. Saukko, B. Knight, Diatoms and the diagnosis of drowning, in: Forensic Pathology, third ed., Hodder Arnold, London, 2004pp.406–408. [12] D.L. Costa, J.R. Lehmann, W.M. Harold, R.T. Drew, Transoral tracheal intubation of rodents using a fiberoptic laryngoscope, Lab. Anim. Sci. 36 (1986) 256–261. [13] O. Elidemir, L.L. Fan, G.N. Colasurdo, A novel diagnostic method for pulmonary aspiration in a murine model: immunocytochemical staining of milk proteins in alveolar macrophage, Am. J. Respir. Crit. Care Med. 161 (2000) 622–626. [14] T. Bjanowski, B. Brinkmann, A.M. Stefanec, R.H. Barckhaus, G. Fechner, Detection and analysis of tracers in experimental drowning, Int. J. Legal Med. 111 (1998) 57–61.