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\def\RunningAuthor{Anitha Roy et al.}
\firstPage{5701}
\articleType{Original Article}
\receivedDate{01 Jun 2020}
\acceptedDate{13 Jul 2020}
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\journalVolume{2020, 11}
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\journalDoi{ijrps.v11i4.3213}
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\title{\textbf{Effectiveness of amikacin administered by autoinjector compared to manual injection on infected excision wound model of Wistar rats}}
\author{Anitha~Roy\textsuperscript{*}\textsuperscript{1},  
            Geetha~R~V\textsuperscript{2},  
            Anitha~Magesh\textsuperscript{3},  
            Senthilkumar~Sivanesan\textsuperscript{3}, 
            Vijayaraghavan~R\textsuperscript{3}~\\[5pt]{\textsuperscript{1}Department of Pharmacology, Saveetha Dental College, SIMATS, Tamil Nadu, India}~\\{\textsuperscript{2}Department of Microbiology, Saveetha Dental College, SIMATS, Tamil Nadu, India}~\\{\textsuperscript{3}Department of Research and Development, Saveetha Medical College, SIMATS, Tamil Nadu, India}}

\begin{abstract}
To evaluate the effectiveness of amikacin administered by autoinjector compared to manual injection on infected excision wound model of Wistar rats. Randomly bred 14 Wistar rats of either sex weighing 180 to 230 g were used for the present study. The study has the approval of the Institutional Animal Ethical Committee. \textit{Pseudomonas aeruginosa } was used for infecting the wounds. 1 mL of  blood was withdrawn aseptically from the orbital sinus under isoflurane anaesthesia, and the biochemical parameters were carried out. All results were expressed as mean \ensuremath{\pm} SEM, and the results were compared statistically by one-way ANOVA using Sigma Plot 13. P-value \textless\ 0.05 was considered statistically significant. The biochemical parameters in the study was more or less similar. The infected rats treated with amikacin showed faster wound contraction compared to control. This study concludes the effectiveness of amikacin administered through autoinjectors and manual injection in infected excision wound model as similar. Therefore, amikacin autoinjector is a better choice to manual injection to overcome from wound infections if it is administered at the right time in case of emergency or whenever required. Injury and wound infection are common in natural and manmade disasters. Serious bacterial wound infections are a potential threat to open injuries. As accessibility to the primary health centre or hospital may not be easy or possible during disasters and to overcome such a situation, an antibacterial autoinjector would be useful.
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Amikacin,\newline Wound infection,\newline autoinjector,\newline Excision
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Anitha~Roy~\\ Phone:\ ~\\  Email:\ anitharoy2015@gmail.com
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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\section{Introduction}
Injury and wound infection are common in  natural and manmade disasters. Serious bacterial wound infections are a potential threat to open injuries. These bacterial infections may cause tetanus or gas gangrene which in  turn may  end up in long  term disabilities like a  chronic wound, bone infection and even death. These infections are of great concern when injured persons present late for definitive care. In disasters, the injured  survivors may exceed available trauma care capacity. Timely and appropriate intervention with an antibiotic is  mandatory to avoid  complications and further morbidity and mortality. As  accessibility to  the primary health centre or hospital may not be  easy or possible during disasters and thus getting appropriate expert care and antibiotic support will be  a question. So, to overcome such a situation, an antibacterial autoinjector would be useful \unskip~\citep{870957:20230920}.


\begin{table*}[!htbp]
\caption{\boldmath {Biochemical parameters estimated in control group and infected wound models treated with amikacin autoinjector and manual injection groups.} }
\label{tw-3356020729a3}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
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\begin{tabulary}{\linewidth}{p{\dimexpr.09700000000000001\linewidth-2\tabcolsep}p{\dimexpr.205\linewidth-2\tabcolsep}p{\dimexpr.1675\linewidth-2\tabcolsep}p{\dimexpr.1705\linewidth-2\tabcolsep}p{\dimexpr.16\linewidth-2\tabcolsep}p{\dimexpr.20\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}S.No & Parameter & Control & Autoinjector & Manual & Statistical \mbox{}\protect\newline Analysis\\
\tblmidrule 
1 &
  Total Protein \mbox{}\protect\newline (g/dL) &
  6.75 \ensuremath{\pm}0.10 &
  7.13 \ensuremath{\pm} 0.38 &
  6.45 \ensuremath{\pm} 0.14 &
  F 1.389 \mbox{}\protect\newline P 0.290\\
2 &
  Albumin \mbox{}\protect\newline (g/dL) &
  3 .98 \ensuremath{\pm} 0.33 &
  3.45 \ensuremath{\pm} 0.24 &
  3.60 \ensuremath{\pm} 0.29 &
  F 0.942 \mbox{}\protect\newline P 0.419\\
3 &
  Globulin \mbox{}\protect\newline ( g/dL) &
  2.78 \ensuremath{\pm} 0.24 &
  3.68 \ensuremath{\pm} 0.41 &
  3.24 \ensuremath{\pm} 0.28 &
  F 1.686 \mbox{}\protect\newline P 0.230\\
4 &
  A/G ratio &
  1.50 \ensuremath{\pm} 0.28 &
  1.02 \ensuremath{\pm} 0.17 &
  1.13 \ensuremath{\pm} 0.10 &
  F 1.603 \mbox{}\protect\newline P 0.245\\
5 &
  C-Reactive \mbox{}\protect\newline Protein &
  0.67 \ensuremath{\pm} 0.11 &
  0.76 \ensuremath{\pm} 0.08 &
  0.79 \ensuremath{\pm} 0.125 &
  F 0.322 \mbox{}\protect\newline P 0.731\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{Values aremean \ensuremath{\pm} SE (n= 4 in  control andmanual  injection}
    
\item{group and n=6 in autoinjector group)}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Percentage of wound contraction in control group and infected wound models treated with amikacin autoinjector and manual injection groups.} }
\label{tw-78f4f273a8c5}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{LLLLLL}
\tbltoprule \rowcolor{kwdboxcolor}S. No & Parameter & Control (Wound \mbox{}\protect\newline contraction \%) & Autoinjector (Wound \mbox{}\protect\newline contraction \%) & Manual (Wound \mbox{}\protect\newline contraction \%) & Statistical Analysis\\
\tblmidrule 
1 &
  4th day &
  0.64 \ensuremath{\pm} 0.14 &
  0.83 \ensuremath{\pm} 0.04 &
  0.84 \ensuremath{\pm} 0.03 &
  P = 0.153\\
2 &
  8th day &
  10.86 \ensuremath{\pm} 0.09 &
  65.60 \ensuremath{\pm} 0.73* &
  63.93 \ensuremath{\pm} 0.72 &
  P \textless\ 0.001\\
3 &
  12th day &
  21.10 \ensuremath{\pm} 0.41 &
  83.64 \ensuremath{\pm} 0.44* &
  80.61 \ensuremath{\pm} 0.29 &
  P \textless\ 0.001\\
4 &
  16th day &
  32.88 \ensuremath{\pm} 0.11 &
  92.67\ensuremath{\pm} 0.45* &
  91.13 \ensuremath{\pm} 0.36 &
  P \textless\ 0.001\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{Statistical Analysis was done with One Way ANOV A with Dunnett's Test P \textless\ 0.05 was considered statistically significant where  n =4 in  control and manual injection  and n = 6 in autoinjector . Values are mean SE* indicates statistical significance compared to control.}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\bgroup
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\caption{\boldmath {Representative photo of excision wound in rat inoculated with \textit{Pseudomonas aeruginosa}}}
\label{f-2730b2ab939d}
\end{figure}
\egroup

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\caption{\boldmath {Rats with wound Infection in metabolic cage}}
\label{f-84aa7ec0a3b7}
\end{figure}
\egroup

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\centering \makeatletter\IfFileExists{images/3f9e9172-04ec-4aa6-993e-73664fa56654-upicture3.png}{\includegraphics{images/3f9e9172-04ec-4aa6-993e-73664fa56654-upicture3.png}}{}
\makeatother 
\caption{\boldmath {Representative photo of wound contraction in rat on day 0,4\ensuremath{^{th}} ,8\ensuremath{^{th}} 12\ensuremath{^{th}} and 16 \ensuremath{^{th }} day of wound infection of control, autoinjector and Manual injection (left to right)}}
\label{f-f79251ed56ca}
\end{figure*}
\egroup
The wound is  a loss  of cellular and functional continuity of living tissues. Wound healing has got different phases in  a sequence {\textendash} haemostasis, inflammation, tissue formation, and tissue remodelling \unskip~\citep{870957:20193770,870957:20193775}. Haemostasis is  characterised by vasoconstriction and blood clotting. This prevents blood loss  and provides a provisional matrix for cell migration. There will be  some complex interaction of cells in  the epidermis, dermis and simultaneous release of  mediators from inflammatory cells, fibroblast and keratinocytes. There will be  the formation of growth factors by local and migratory cells which stimulate migration of  fibroblasts into the wounded area, which proliferate to form an extracellular matrix. The migration and proliferation of  keratinocytes continue until the wound get entirely covered. Another vital process involved is  angiogenesis which involves migration, proliferation and organisation of vascular endothelial cells. The matrix formation and epithelialisation depend on angiogenesis. Complete healing occurs only after knitting the wounded surface firmly by collagen \unskip~\citep{870957:20193774}. The wound can occur due to severe burns, tissue damage and trauma or in  diseases like  diabetes. Whatever be  the reason behind the wound, complete and rapid healing is  critical during  medical interventions.

Wound infections are widespread in  developing countries than in developed countries \unskip~\citep{870957:20193769}. Infection can slow down wound healing, even though it is  a natural process \unskip~\citep{870957:20193781}. The clinical signs and symptoms of infection include pain, erythema, oedema, heat, purulent exudates, serous exudate with concurrent inflammation, delayed healing, discolouration of granulation tissue, friable granulation tissue, pocketing at the base, foul odour and wound breakdown \unskip~\citep{870957:20193779}.\textit{ Pseudomonas } and \textit{Staphylococcus } are major  organisms often isolated from infected wounds. They are capable of forming a biofilm in  the wound surface, and they produce an enzyme {\textendash} protease responsible for the degradation of the extracellular matrix essential for wound healing.

Amikacin is an aminoglycoside antibiotic highly effective against gram-negative organism like \textit{Pseudomonas}. It inhibits bacterial protein synthesis and produces a bactericidal effect. Hence, the bactericidal effect of the drug can reduce the burden of microorganism and reduction in biofilm formation and thereby can hasten wound healing. This study was carried out to show the effectiveness of amikacin autoinjector compared with manual injection in wound infection.
    
\section{Materials and Methods}
\textbf{Animals}

Randomly bred 14 Wistar rats of either sex weighing 180 to 230 g were used for the present study. The animals were housed and maintained in polypropylene cages in Centre for Laboratory and Animal Research (CLAR), Saveetha University as per the guidelines of Committee for Control and Supervision of Experiments on Animals (CPCSEA). They were fed with drinking water and commercially available pellets (VRK Nutritional Solutions, Chennai India) ad libitum. The study has the approval of the Institutional Animal Ethical Committee. 

\textbf{Bacteria for wound infection}

\textit{Pseudomonas aeruginosa }was obtained from the Department of Microbiology, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences (Chennai, India) for the study. The organism was maintained in nutrient agar slant (Hi-Media Pvt. Ltd., India) at 4\ensuremath{^\circ}C.

\textbf{Excision wound model and infection}

The animals were randomly allocated into three groups. Group1 served as a control group with four animals. Group 2, with six animals on amikacin autoinjector and group 3 with four animals on amikacin manual injection. Since amikacin autoinjector is  a new device, more animals were used for the wound infection study. Excision wound model was used for this study. A pre-decided area in  the dorsal surface of the rats was shaved, and wounds measuring around 500 mm\ensuremath{^{2}}\unskip~\citep{870957:20193773} were created using  a sterile surgical blade under isofluorane anaesthesia aseptically. The wound was infected by instilling 100 \ensuremath{\mu}l of \textit{Pseudomonas aeruginosa } using a micropipette in a biosafety cabinet(Type 2) and the animals were observed for 4 hours (Figure~\ref{f-2730b2ab939d}). The rats were then housed in  separate metabolic cages to  develop infection (Figure~\ref{f-84aa7ec0a3b7}).

\textbf{Drug administration}

The infected animals in  Group 1 were kept as control. Group 2 and group 3 were administered with 63 mg/mL of amikacin i.p  from the fourth day, by autoinjector and manual injection (1.2 mL) continuously for four days. 

\textbf{Rate of wound contraction}

Wound size was measured every four days interval (4\ensuremath{^{th}}, 8\ensuremath{^{th}}, 12\ensuremath{^{th}} and 16\ensuremath{^{th}} day) with the help of butter paper and graph paper and the percentage of wound the contraction was calculated using the following formula (Figure~\ref{f-f79251ed56ca}). The percentage of wound contraction was calculated by dividing the difference in wound area of a particular day from the zero-day by zero-day area and then multiplying it with hundred \unskip~\citep{870957:20193777}. 

\textbf{Blood sample collection}

On the 8\ensuremath{^{th}} day, 1 mL of blood was withdrawn aseptically from the orbital sinus under isoflurane anaesthesia, and the biochemical parameters were carried out. After the withdrawal of blood, the animals were returned to the metabolic cages and observed for wound contraction.

\textbf{The blood biochemical parameters}

Total protein, albumin, globulin and C-reactive protein (CRP) were estimated using autoanalyser. The turbidometric method was used for C-reactive protein.

\textbf{Statistical analysis}

All results were expressed as mean \ensuremath{\pm} SEM, and the results were compared statistically by one-way ANOVA using Sigma Plot 13. P-value \textless\ 0.05 was considered statistically significant.
    
\section{Results and Discussion}
Biochemical parameters like  total protein, albumin,  globulin and C-reactive protein was analysed. Total protein was 6.75 \ensuremath{\pm} 0.10 g/dL for control. Though there was little increase in  the autoinjector group, 7.13 \ensuremath{\pm} 0.38 g/dL it  was not statistically significant. In manual injection, the total protein was 6.45 \ensuremath{\pm} 0.14 g/dL. The albumin level were 3 .98 \ensuremath{\pm} 0.33 g/dL, 3.45 \ensuremath{\pm} 0.24 g/dL and 3.60 \ensuremath{\pm} 0.29 g/dL in  the control, autoinjector and manual group respectively. The globulin also was similarly with a slight increase in the  autoinjector group but not clinically significant. The globulin level was 2.78 \ensuremath{\pm} 0.24 g/dL, 3.68 \ensuremath{\pm} 0.41g/dL and 3.23 \ensuremath{\pm} 0.28 g/dL; the albumin- globulin ratio was 1.50 \ensuremath{\pm} 0.28, 1.023 \ensuremath{\pm} 0.17 and 1.13 \ensuremath{\pm} 0.10 for the control, autoinjector group and manual respectively. The C-reactive protein was 0.67 \ensuremath{\pm} 0.11 mg/dL for control group and for autoinjector and manual injection group, it  was 0.76 \ensuremath{\pm} 0.08 mg/dL and 0.79 \ensuremath{\pm} 0.13 mg/dL respectively (Table~\ref{tw-3356020729a3}).

The wound in  all three groups was almost the same in  the beginning. The wound closure on the fourth day was 0.64 \ensuremath{\pm} 0.14 \%, 0.83 \ensuremath{\pm} 0.04\% and 0.84 \ensuremath{\pm} 0.03\% respectively, for the control, autoinjectors and manual injection group. There was a significant increase in  healing (P \textless\ 0.001) on day eight and the wound closure was 10.86 \ensuremath{\pm} 0.09 \%, 65.60 \ensuremath{\pm}   0.73 \% and 63.93 \ensuremath{\pm} 0.72 \% for control, autoinjector and manual injection, respectively. On day 12 still better-wound closure was observed with 21.100 \ensuremath{\pm} 0.41 \%, 83.64 \ensuremath{\pm} 0.44 \% and 80.61 \ensuremath{\pm} 0.29\% for control, autoinjectors and manual injection, respectively (P \textless\ 0.001). On day 16 the wound closure was 32.88 \ensuremath{\pm} 0.11 \%, 92.67 \ensuremath{\pm} 0.45 \% and 91.13 \ensuremath{\pm} 0.36 \% for control, autoinjectors and manual respectively with a high significance from control (0.001) (Table~\ref{tw-78f4f273a8c5} and Figure~\ref{f-f79251ed56ca}).

Rat models are excellent for skin wound healing study as it  can be  used in  terms of size, shape, type and depth of wound injury. They are selected for skin wound healing model because of their small size, ready availability and economical with limited housing facility. Both incision and excision wound models can be  prepared to study the healing effect on the dorsum of the animal because they  will not allow  the animal to  reach and manipulate the wound. The skin of rat and humans have a more or less  similar pattern of the epidermis, basement membrane and hair follicle and dermis. In this study,  the excision wound model was used.

During any injury, the body responds by restoring tissue injury by the synthesis of a connective tissue matrix at the site of the wound. The wound is strengthened by the fibrous protein, collagen, which is the major component of the extracellular matrix. The increase in hydroxyl proline content supports the increased migration of fibroblast cells, epithelial cells and collagen for the synthesis of the extracellular matrix. A decrease in hydroxyl proline can prolong the inflammatory phase of wound healing and then inhibits both epithelial regeneration and proliferation of fibroblast. Wound contraction is a necessary feature for the healing process \unskip~\citep{870957:20193771}. 

Contaminated wounds, penetrating wounds, abdominal trauma, compound fractures, lacerations greater than 5 cm,  wounds with devitalised tissue, high-risk anatomical sites like  hand or foot are at  high risk to become infected. Antibiotic prophylaxis is  indicated in  Injuries requiring surgical intervention within 2 hr. Injury and wound infections are quite common in  natural and manmade disasters. Hence, in  this study, wound infection with \textit{Pseudomonas } was created to check the effectiveness of amikacin autoinjector in  comparison to manual injection as it is  one of the organisms often isolated from infected wounds. They produce an enzyme {\textendash} \textit{protease } responsible for the  degradation of the extracellular matrix essential for wound healing and thus can delay wound healing. Amikacin is  effective against gram-negative organism like \textit{Pseudomonas}. The bactericidal effect of amikacin might have reduced the burden of  microorganism and reduction in  biofilm formation and  thereby could hasten wound healing. On the other hand, the delayed wound healing in  the infected control group may be  due to the presence of microorganism and their metabolites \unskip~\citep{870957:20193769}.

The wound closure was slow in  the beginning, and once the animal showed a response to the antibacterial agent, the closure rate was faster in  the treated group compared to control. The wound closure was significant from day eight, and the wound closure rate was faster in  the treated group compared to control. More  than 90\% closure of the wound was found on day 16 in amikacin treated groups. Control group took more than 22 days to get completely closed. The antibacterial activity of the antibiotic could substantially reduce the burden of the wound pathogen leading to complete and proper wound healing.

The animal weight, food and water intake were monitored regularly. The wound in the untreated group was wet and with malodour due to infection. There could be a link between malodour and delayed wound healing. Malodour was reduced in the treated group, which may be due to a reduction in microbial presence due to antibiotic therapy and the restart of healing \unskip~\citep{870957:20193780,870957:20193776}.

Albumin is a gross indicator of nutritional and fluid status. It is needed for tissue synthesis and fighting against infection. It serves as building blocks of all cells and body tissues along with globulin and total protein. Globulin was slightly increased in the autoinjector group but was not significantly high to indicate the superiority of the treatment. Systemic infection may have a more profound effect. Since the wound is topical, changes could not be detected.

C- reactive protein (CRP) is commonly used as a diagnostic measure to identify wounds which require antimicrobial therapy (Kingsley and Jones, 2008). It is a marker for acute inflammation. It is reasonable to presume that CRP levels may get elevated because of infection and fall in response to effective antimicrobial therapy. Many acute inflammatory conditions are diagnosed with its estimation alone or with ESR, WBC count \unskip~\citep{870957:20193772}. In response to acute and chronic inflammatory events, it will be secreted from macrophages, adipocytes and hepatocytes. Persistently elevated CRP is seen in non-healing chronic wounds \unskip~\citep{870957:20193778}. Within 24-48 hrs, the changes in CRP levels can occur in response to the presence or withdrawal of the stimulation, such as a bacterial infection. The biochemical parameters were not significant from control as the infection was not a systemic one.The effect of amikacin autoinjector alone \unskip~\citep{870957:20231043} and in combination with cefazolin \unskip~\citep{870957:20231044} was tested for biochemical and oxidative stress parameters and found to be similar with manual injection.
    
\section{Conclusions}
The infected rats treated with amikacin showed faster wound contraction compared to  control. This study concludes the effectiveness of  amikacin administered through autoinjectors and manual injection in  infected excision wound model as similar. Therefore, amikacin autoinjector is  a better choice to manual injection to  overcome from wound infections if it is  administered at the right time in  case of emergency or whenever required.

\textbf{Acknowledgement}

The authors thank the university setting for all the facilities provided to carry out the research.

\textbf{Conflict of interest}

The authors declare that they have no conflict of interest for this study.

\textbf{Funding agency}

Self-funded.
    

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