Violent political conflict is on the front pages, in Iraq, Afghanistan, and Sudan. This provocative piece discusses lessons we can learn from past conflicts in dealing with future ones

Violent political conflict, and its impact, is again on the front pages—in Iraq, Afghanistan, and Sudan. While the situation in Darfur is now particularly urgent (see sidebar) [ 1 , 2 , 3 , 4 , 5 ], there are many other settings in which complex political emergencies are undermining health service provision and threatening human rights. Such emergencies have a direct impact on health (see  Table 1 ). They also impair the functioning of health systems through, for example, destruction of infrastructure (such as clinics and vehicles), reduced access to medicines, death of health workers, and weakened national capacity for health policy-making [ 6 ].Examples of the Direct Impact of Conflict on Health
Adapted from [ 7 ], with permission from the World Health OrganizationSuch violent political conflicts stir us—the global health community—to discover our own humanity amidst the bloodshed. How best should we respond? Despite unique features in each setting, we must learn lessons from previous conflicts to help guide our response to current and future ones. There are six key lessons that emerge from studying health in conflict settings.Lessons from Conflict Settings
 Violent conflict is driven by politics and economics [ 7 ].  Complex political emergencies (1) occur within and across state boundaries, (2) have political antecedents typically relating to competition for power and resources, (3) are protracted in duration, (4) express existing social, political, economic, and cultural structures and cleavages, and (5) are often characterised by one sector preying on other parts of the community [ 8 ]. Damage to health is not just a side effect but may be the objective for violent groups. In complex political emergencies, we can typically identify three groups: the winners, the ‘conflict entrepreneurs’ (who seek the perpetuation of conflict because they profit economically or politically), and the losers, whose lives and livelihoods are imperilled. Humanitarian and relief agencies increasingly recognise that belligerents may seek to control or manipulate the inflow of humanitarian and relief resources [ 9 ]. A political economy perspective helps identify those interests, which may impede the transition to peace [ 7 ]. Sudan—Conflict and Health The current crisis in Darfur reflects a devastatingly acute episode in the chronic internal conflict that has plagued Sudan since 1983. The cost of this conflict has been enormous: over 2 million lives lost, over 628,000 refugees from Sudan in neighbouring countries, and over 4 million people internally displaced [ 1 ]. In southern Sudan, the conflict has led to widespread ill health and has severely compromised the well-being of women and children. Indicators of immunisation, nutrition, primary school completion, and antenatal care are among the worst in the world. About 95,000 children under five years old died last year, most from preventable disease [ 2 ]. Statistics from UNICEF are chilling: ‘A girl born in southern Sudan has a better chance of dying in pregnancy or childbirth than of completing primary school….One in nine women dies in pregnancy or childbirth but only one in a hundred girls completes primary school’ [ 2 ]. Communities in Darfur face ongoing violence from militia supported by the government of Sudan. The fighting has resulted in large-scale destruction of villages, rape, and kidnapping. About 15,000–30,000 lives are estimated to have been lost from January 2003 to June 2004 [ 3 ]. Surveys by Médecins Sans Frontières found death rates of three to five per 10,000 people/day in Mornay and Zalinge villages (the emergency threshold level is set at one death per 10,000/day) [ 4 ]. Over 300,000 people are at risk if humanitarian access remains restricted. Of displaced Darfurians, 90% need shelter and latrines, and over half lack access to primary health care [ 3 ]. Food insecurity is widespread and is being used as a ‘weapon of war’ [ 5 ] resulting in widespread nutritional problems. Despite widespread concern, information gaps abound, and humanitarian agencies report having access to only a fraction of those most affected. Yet, genocide is taking place in real time.
 Appreciating context is crucial.  The nature of the conflict—its background, history, and the different forms of violence involved—will greatly influence health outcomes. Most conflicts are today intra-national rather than international [ 10 ]. Internal conflicts affect populations through forced migration, violence, and human rights abuses including torture, disappearances, and rape. The forms of violence and types of health damage relate to the phase of the conflict, the sophistication of weapons used, the degree of involvement of regular military forces, the extent of terrorism employed, and the extent to which genocide is intended. Ongoing insecurity and instability may be present even after the ostensible end to the conflict, as in latter-day Afghanistan and Iraq. Challenges to governance, to service delivery, and to the reestablishment of livelihoods may persist for years. A 2003 survey in Iraq found that despite the brief duration of the war and the intent to spare hospitals and clinics from direct attack, many people suffered in the post-war period, primarily as a result of disruption to civil order [ 11 ]. Recent reports highlight the difficulties of re-establishing the health system in Iraq—partly because of a failure to appreciate the cultural and health services context [ 12 ].
 Better care can save lives.  Emergency relief efforts are increasingly based upon empirical evidence, and priority health issues are much more effectively addressed than previously. Emphasis is typically placed upon disease surveillance, immunisation, control of infectious diseases, reproductive health, water and sanitation, shelter, and nutrition [ 13 ]. Mental health, sexually transmitted infections, and HIV have recently attracted additional attention. Standards have improved, can be further improved, and warrant widespread dissemination and application. The more-established humanitarian agencies have accepted that their relief efforts must be as evidence-based as possible. This principle should also apply to the post-conflict period, during which the health of affected communities continues to suffer [ 14 ].
 About 70% of structures were destroyed in Dili, East Timor, in the violence wrought by Indonesian militia after the referendum in 1999 (Photo: Anthony Zwi.)
 We need enhanced accountability for humanitarian action.  Despite a developing evidence base for health-related humanitarian action, evaluations of humanitarian activities have found ongoing problems. These include poor standards of delivery, duplication of efforts by different agencies, lack of coordination, and failing to learn from prior experience. The Sphere Project has advocated minimum standards for the delivery of humanitarian assistance, and has established a “Humanitarian Charter” ( http://www.sphereproject.org ). The project's objectives and achievements have been to improve the quality of humanitarian action and promote a movement concerned with the rights and dignity of those caught up in war and disaster [ 15 , 16 ]. The Active Learning Network for Accountability and Performance in Humanitarian Action ( http://www.alnap.org ) seeks to ensure that lessons are learned, distilled, and disseminated. At a meeting in Stockholm in June 2003, key international donors committed themselves to ‘good humanitarian donorship’, which recognises the importance of promoting standards in humanitarian action [ 17 ]. However, recent sober reflection suggests that donors and humanitarian agencies could do better: ‘An ailing humanitarian enterprise is labouring under pressures from the external environment over which it has little control, while struggling with issues internal to its own function for which it should take greater responsibility’ [ 18 ].
 Militarization of humanitarian efforts is problematic.  Multinational military forces have played a major part in recent conflicts in Kosovo, East Timor, Sierra Leone, Iraq, and Afghanistan. The military has become increasingly involved not only in waging war but also in seeking to win the peace; it is increasingly active in delivering emergency relief. It not only provides services—sometimes necessary to deliver needed relief—but also seeks to ‘win hearts and minds’ while operating within structures responsive to military and foreign policy directives. The result has seen a blurring of the separation between military and humanitarian efforts [ 19 ]. This can make humanitarian agencies a target—recent examples include the bombing of United Nations headquarters and the International Committee of the Red Cross in Iraq and the recent, reluctant withdrawal of Médecins Sans Frontières from Afghanistan following the murder of five aid workers [ 20 ]. Emerging evidence and good practice in civil-military cooperation highlights the importance of (1) promoting needs-based assistance free of discrimination, (2) civilian-military distinction in humanitarian action, (3) independence of humanitarian organisations from political pressures and interference, and (4) the security of humanitarian personnel [ 19 ].
 The transition from emergency relief to development is poorly managed.  The objectives of humanitarian relief activity (saving lives and livelihoods) differ from those of development (building sustainable systems, promoting equity, building systems of governance, and eradicating poverty). In each phase there are different actors, strategies, and approaches. The increasing politicisation of humanitarian intervention [ 21 , 22 ] brings threats and dangers, undermining key humanitarian principles. The balance between relief and development will vary over time and place; getting the balance right and adequately resourcing the transition warrants careful research, documentation, reflection, and the commitment of appropriate longer-term funding.
Lessons from Conflict Settings
Violent conflict is driven by politics and economics [ 7 ].  Complex political emergencies (1) occur within and across state boundaries, (2) have political antecedents typically relating to competition for power and resources, (3) are protracted in duration, (4) express existing social, political, economic, and cultural structures and cleavages, and (5) are often characterised by one sector preying on other parts of the community [ 8 ]. Damage to health is not just a side effect but may be the objective for violent groups. In complex political emergencies, we can typically identify three groups: the winners, the ‘conflict entrepreneurs’ (who seek the perpetuation of conflict because they profit economically or politically), and the losers, whose lives and livelihoods are imperilled. Humanitarian and relief agencies increasingly recognise that belligerents may seek to control or manipulate the inflow of humanitarian and relief resources [ 9 ]. A political economy perspective helps identify those interests, which may impede the transition to peace [ 7 ].Violent conflict is driven by politics and economics [ 7 ].Sudan—Conflict and Health
The current crisis in Darfur reflects a devastatingly acute episode in the chronic internal conflict that has plagued Sudan since 1983. The cost of this conflict has been enormous: over 2 million lives lost, over 628,000 refugees from Sudan in neighbouring countries, and over 4 million people internally displaced [ 1 ].In southern Sudan, the conflict has led to widespread ill health and has severely compromised the well-being of women and children. Indicators of immunisation, nutrition, primary school completion, and antenatal care are among the worst in the world. About 95,000 children under five years old died last year, most from preventable disease [ 2 ].Statistics from UNICEF are chilling: ‘A girl born in southern Sudan has a better chance of dying in pregnancy or childbirth than of completing primary school….One in nine women dies in pregnancy or childbirth but only one in a hundred girls completes primary school’ [ 2 ].Communities in Darfur face ongoing violence from militia supported by the government of Sudan. The fighting has resulted in large-scale destruction of villages, rape, and kidnapping. About 15,000–30,000 lives are estimated to have been lost from January 2003 to June 2004 [ 3 ]. Surveys by Médecins Sans Frontières found death rates of three to five per 10,000 people/day in Mornay and Zalinge villages (the emergency threshold level is set at one death per 10,000/day) [ 4 ]. Over 300,000 people are at risk if humanitarian access remains restricted. Of displaced Darfurians, 90% need shelter and latrines, and over half lack access to primary health care [ 3 ]. Food insecurity is widespread and is being used as a ‘weapon of war’ [ 5 ] resulting in widespread nutritional problems.Despite widespread concern, information gaps abound, and humanitarian agencies report having access to only a fraction of those most affected. Yet, genocide is taking place in real time.Appreciating context is crucial.  The nature of the conflict—its background, history, and the different forms of violence involved—will greatly influence health outcomes. Most conflicts are today intra-national rather than international [ 10 ]. Internal conflicts affect populations through forced migration, violence, and human rights abuses including torture, disappearances, and rape. The forms of violence and types of health damage relate to the phase of the conflict, the sophistication of weapons used, the degree of involvement of regular military forces, the extent of terrorism employed, and the extent to which genocide is intended. Ongoing insecurity and instability may be present even after the ostensible end to the conflict, as in latter-day Afghanistan and Iraq. Challenges to governance, to service delivery, and to the reestablishment of livelihoods may persist for years. A 2003 survey in Iraq found that despite the brief duration of the war and the intent to spare hospitals and clinics from direct attack, many people suffered in the post-war period, primarily as a result of disruption to civil order [ 11 ]. Recent reports highlight the difficulties of re-establishing the health system in Iraq—partly because of a failure to appreciate the cultural and health services context [ 12 ].Appreciating context is crucial.Better care can save lives.  Emergency relief efforts are increasingly based upon empirical evidence, and priority health issues are much more effectively addressed than previously. Emphasis is typically placed upon disease surveillance, immunisation, control of infectious diseases, reproductive health, water and sanitation, shelter, and nutrition [ 13 ]. Mental health, sexually transmitted infections, and HIV have recently attracted additional attention. Standards have improved, can be further improved, and warrant widespread dissemination and application. The more-established humanitarian agencies have accepted that their relief efforts must be as evidence-based as possible. This principle should also apply to the post-conflict period, during which the health of affected communities continues to suffer [ 14 ].Better care can save lives.We need enhanced accountability for humanitarian action.  Despite a developing evidence base for health-related humanitarian action, evaluations of humanitarian activities have found ongoing problems. These include poor standards of delivery, duplication of efforts by different agencies, lack of coordination, and failing to learn from prior experience. The Sphere Project has advocated minimum standards for the delivery of humanitarian assistance, and has established a “Humanitarian Charter” ( http://www.sphereproject.org ). The project's objectives and achievements have been to improve the quality of humanitarian action and promote a movement concerned with the rights and dignity of those caught up in war and disaster [ 15 , 16 ]. The Active Learning Network for Accountability and Performance in Humanitarian Action ( http://www.alnap.org ) seeks to ensure that lessons are learned, distilled, and disseminated. At a meeting in Stockholm in June 2003, key international donors committed themselves to ‘good humanitarian donorship’, which recognises the importance of promoting standards in humanitarian action [ 17 ]. However, recent sober reflection suggests that donors and humanitarian agencies could do better: ‘An ailing humanitarian enterprise is labouring under pressures from the external environment over which it has little control, while struggling with issues internal to its own function for which it should take greater responsibility’ [ 18 ].We need enhanced accountability for humanitarian action.Militarization of humanitarian efforts is problematic.  Multinational military forces have played a major part in recent conflicts in Kosovo, East Timor, Sierra Leone, Iraq, and Afghanistan. The military has become increasingly involved not only in waging war but also in seeking to win the peace; it is increasingly active in delivering emergency relief. It not only provides services—sometimes necessary to deliver needed relief—but also seeks to ‘win hearts and minds’ while operating within structures responsive to military and foreign policy directives. The result has seen a blurring of the separation between military and humanitarian efforts [ 19 ]. This can make humanitarian agencies a target—recent examples include the bombing of United Nations headquarters and the International Committee of the Red Cross in Iraq and the recent, reluctant withdrawal of Médecins Sans Frontières from Afghanistan following the murder of five aid workers [ 20 ]. Emerging evidence and good practice in civil-military cooperation highlights the importance of (1) promoting needs-based assistance free of discrimination, (2) civilian-military distinction in humanitarian action, (3) independence of humanitarian organisations from political pressures and interference, and (4) the security of humanitarian personnel [ 19 ].Militarization of humanitarian efforts is problematic.The transition from emergency relief to development is poorly managed.  The objectives of humanitarian relief activity (saving lives and livelihoods) differ from those of development (building sustainable systems, promoting equity, building systems of governance, and eradicating poverty). In each phase there are different actors, strategies, and approaches. The increasing politicisation of humanitarian intervention [ 21 , 22 ] brings threats and dangers, undermining key humanitarian principles. The balance between relief and development will vary over time and place; getting the balance right and adequately resourcing the transition warrants careful research, documentation, reflection, and the commitment of appropriate longer-term funding.The transition from emergency relief to development is poorly managed.What Gaps Remain in Our Knowledge? Despite the knowledge we have gained on responding to violent political conflict, many important gaps remain.
 In southern Sudan, the conflict has affected the well-being of women and girls (Illustration: Margaret Shear, Public Library of Science.) We still do not hear the voices of those most affected or of the service providers seeking to assist. The reality of people's experiences is inadequately appreciated [ 23 ]; whatever we learn of their fears, challenges, and suffering is typically represented and reported through sanitised language and media. The language used dehumanises ‘the enemy’ and blunts our senses to the reality of atrocity and to the negative effects of our own countries’ interventions. Within the health sector, ensuring that we hear the voices of service providers and carers will help bring home the reality of system disruption, destruction, and damage and will simultaneously document the mechanisms and potential for effective responses. The new communication technologies provide immense opportunity to ensure that experience is placed in the public domain from where lessons can be drawn and better practice promoted. We know little about how communities and systems survive adversity. In most settings, the inherent ability and ingenuity of people and systems allows them to withstand instability and insecurity. Health personnel and health systems could play a valuable role in these fragile settings—assisting individuals, communities, and systems to further develop their coping strategies, adaptations, and responses. But whether health systems do so and how is unclear. Failing to support and maintain these systems may result in much greater challenges when we seek at a later stage to resuscitate them. We also know relatively little about whether the health sector can indeed make a special contribution to building the peace. While it has been forcefully argued that the health sector is uniquely placed to play a role in peace building [ 24 ], the evidence for this remains limited [ 25 ]. We know little about how health workers see and respond to these challenging roles. The health sector could play a role in demonstrating the values and priorities of government, reflecting the relationship between those with and without resources, and the relationship between those who do and do not have protection. In the aftermath of major periods of violence, the health sector could also help to ensure that the structural inequities that preceded the violence and may have contributed to it, are not reinforced and the same injustices not recreated. But, engagement around health is not always positive: the health system is open to abuse and has been abused by repressive systems.
What Gaps Remain in Our Knowledge?
Despite the knowledge we have gained on responding to violent political conflict, many important gaps remain.We still do not hear the voices of those most affected or of the service providers seeking to assist. The reality of people's experiences is inadequately appreciated [ 23 ]; whatever we learn of their fears, challenges, and suffering is typically represented and reported through sanitised language and media. The language used dehumanises ‘the enemy’ and blunts our senses to the reality of atrocity and to the negative effects of our own countries’ interventions. Within the health sector, ensuring that we hear the voices of service providers and carers will help bring home the reality of system disruption, destruction, and damage and will simultaneously document the mechanisms and potential for effective responses. The new communication technologies provide immense opportunity to ensure that experience is placed in the public domain from where lessons can be drawn and better practice promoted.We know little about how communities and systems survive adversity. In most settings, the inherent ability and ingenuity of people and systems allows them to withstand instability and insecurity. Health personnel and health systems could play a valuable role in these fragile settings—assisting individuals, communities, and systems to further develop their coping strategies, adaptations, and responses. But whether health systems do so and how is unclear. Failing to support and maintain these systems may result in much greater challenges when we seek at a later stage to resuscitate them.We also know relatively little about whether the health sector can indeed make a special contribution to building the peace. While it has been forcefully argued that the health sector is uniquely placed to play a role in peace building [ 24 ], the evidence for this remains limited [ 25 ]. We know little about how health workers see and respond to these challenging roles. The health sector could play a role in demonstrating the values and priorities of government, reflecting the relationship between those with and without resources, and the relationship between those who do and do not have protection. In the aftermath of major periods of violence, the health sector could also help to ensure that the structural inequities that preceded the violence and may have contributed to it, are not reinforced and the same injustices not recreated. But, engagement around health is not always positive: the health system is open to abuse and has been abused by repressive systems.From Learning Lessons to Sound Policy Perhaps the most important gap of all is between observing lessons and putting them into practice. We urgently need to transform evidence and experience into sound policy. We need more sophisticated policy analyses, more sensitive policy-making, and more relevant research. Policy in these difficult areas will never be entirely evidence-based—often it will at best be ‘evidence-informed’. Our objective must be to promote organisations and systems that are able to reflect on experience, work with partners to critically analyse and learn, and thereby formulate better responses. Violent political conflict will continue to challenge the global health community. International policy-makers and funders must support more extensive documentation and reflection: the building blocks of better practice.
From Learning Lessons to Sound Policy
Perhaps the most important gap of all is between observing lessons and putting them into practice. We urgently need to transform evidence and experience into sound policy. We need more sophisticated policy analyses, more sensitive policy-making, and more relevant research. Policy in these difficult areas will never be entirely evidence-based—often it will at best be ‘evidence-informed’. Our objective must be to promote organisations and systems that are able to reflect on experience, work with partners to critically analyse and learn, and thereby formulate better responses. Violent political conflict will continue to challenge the global health community. International policy-makers and funders must support more extensive documentation and reflection: the building blocks of better practice.


Background The molecular events, following ischemia and reperfusion (I/R) of the liver during transplantation are largely unknown. There is evidence that apoptotic and necrotic events may take place, and occasionally result in primary graft dysfunction. We herein report two cases, where significant I/R injury correlated with the development of liver calcification and primary liver dysfunction.
Background
The molecular events, following ischemia and reperfusion (I/R) of the liver during transplantation are largely unknown. There is evidence that apoptotic and necrotic events may take place, and occasionally result in primary graft dysfunction. We herein report two cases, where significant I/R injury correlated with the development of liver calcification and primary liver dysfunction.Case Presentation Both patients with clinical and biochemical evidence of primary graft dysfunction demonstrated calcification at light and electron microscopy levels. In addition, one patient had macroscopic evidence of calcification on cross-sectional imaging. Both patients died secondary to the sequelae of the graft dysfunction.
Case Presentation
Both patients with clinical and biochemical evidence of primary graft dysfunction demonstrated calcification at light and electron microscopy levels. In addition, one patient had macroscopic evidence of calcification on cross-sectional imaging. Both patients died secondary to the sequelae of the graft dysfunction.Conclusions Severe I/R-induced injury to the liver, clinically leads to graft dysfunction. This is due to advanced apoptotic and/or necrotic events at the hepatocyte level that may, on the most severe form, lead to calcification. The study of microcalcification at the early posttransplant period could provide insight in the events taking place following significant ischemia/reperfusion-induced injury to the graft.
Conclusions
Severe I/R-induced injury to the liver, clinically leads to graft dysfunction. This is due to advanced apoptotic and/or necrotic events at the hepatocyte level that may, on the most severe form, lead to calcification. The study of microcalcification at the early posttransplant period could provide insight in the events taking place following significant ischemia/reperfusion-induced injury to the graft.

Background Liver transplantation (LT) remains the only treatment for end-stage liver disease. However the transplant procedure mandates cold perfusion, hypothermic storage, warm ischemia and warm reperfusion of the graft, resulting in Ischemia/Reperfusion (I/R)-induced injury to the transplanted graft. Although the introduction of the University of Wisconsin solution (UW) has improved clinical outcomes, I/R injury remains one of the major clinical problems following LT leading, in cases of marginal donor quality or prolonged cold or warm ischemic time, to the development of graft dysfunction or non function. Recently the cellular events following liver I/R during LT have been brought to sharp focus. Nevertheless, it is still controversial if the major mode of cell death during I/R is apoptosis and/or necrosis [ 1 ]. It has been shown in an animal model of viral-induced hemorrhagic liver necrosis, that the liver undergoes several morphologic changes including mineralization [ 2 ]. Furthermore, previous clinical reports have documented the development of calcifications in liver upon extensive ischemia [ 3 , 4 ]. The above observations may reflect a high degree of cell damage leading to an enhanced apoptotic cell engulfment by non-professional phagocytes of the liver [ 5 ] with subsequent necrosis and biomineralization. In this report, using a combination of cross-sectional imaging, histochemistry, and light (LM) as well as transmission electron microscopy (TEM) we identified liver calcification, in two grafts with severe I/R injury following LT.
Background
Liver transplantation (LT) remains the only treatment for end-stage liver disease. However the transplant procedure mandates cold perfusion, hypothermic storage, warm ischemia and warm reperfusion of the graft, resulting in Ischemia/Reperfusion (I/R)-induced injury to the transplanted graft. Although the introduction of the University of Wisconsin solution (UW) has improved clinical outcomes, I/R injury remains one of the major clinical problems following LT leading, in cases of marginal donor quality or prolonged cold or warm ischemic time, to the development of graft dysfunction or non function.Recently the cellular events following liver I/R during LT have been brought to sharp focus. Nevertheless, it is still controversial if the major mode of cell death during I/R is apoptosis and/or necrosis [ 1 ]. It has been shown in an animal model of viral-induced hemorrhagic liver necrosis, that the liver undergoes several morphologic changes including mineralization [ 2 ]. Furthermore, previous clinical reports have documented the development of calcifications in liver upon extensive ischemia [ 3 , 4 ]. The above observations may reflect a high degree of cell damage leading to an enhanced apoptotic cell engulfment by non-professional phagocytes of the liver [ 5 ] with subsequent necrosis and biomineralization.In this report, using a combination of cross-sectional imaging, histochemistry, and light (LM) as well as transmission electron microscopy (TEM) we identified liver calcification, in two grafts with severe I/R injury following LT.Case Presentations Case 1 A 65-year-old man of Asian origin underwent LT at our institution on June 2002, for decompensated Hepatitis B related cirrhosis. The patient did not have a history of abnormal calcium metabolism, or hyperparathyroidism. Upon listing his serum ionized calcium was 1.07 mmol/L. The donor was a previously healthy, 42-year-old male, who suffered intracranial bleeding following a motor vehicle crash. He did not suffer any period of hypoxia but he was hypotensive (SBP = 90 mm Hg) prior to procurement. His serum liver function tests were normal prior to harvesting (AST 32 U/L, ALT 40 U/L). The organ was procured by our institution's transplant team. During procurement the liver was found to be well perfused with no focal injuries and no macroscopic evidence of steatosis. The recipient underwent an uncomplicated conventional LT without the use of a veno-venous bypass. There were no periods of hypoxia or severe hypotension during transplantation. The cold ischemic time of the graft was 8 hours while the warm ischemic time was 45 minutes. The graft reperfused well and no biopsies were taken. Intravenous methylprednisolone (500 mg) was administered intraoperatively, and postoperatively the patient received induction with Antithymocyte Globulin, which is the protocol followed at our institution. During transplantation the patient received a total of 4 units of packed red blood cells (PRBCs) and 6 units of fresh frozen plasma (FFP). At the time of LT, the international normalization ratio (INR) was 1.53, while serum total bilirubin was 103 μmol/L (Figure  1A,1B ). On postoperative day 2 the patient had a peak of his serum AST (3469 U/L) and at this point he had further biochemical evidence of primary graft dysfunction, with inability to normalize his INR (Figure  1B ), and with progressive elevation of his total serum bilirubin (Figure  1A ). Repeated ultrasonographic examination revealed a patent hepatic artery and portal vein, as well as patent hepatic veins. At this point, a liver biopsy demonstrated severe reperfusion injury with several apoptotic bodies, several dystrophic calcifications (Figure  1E ), and no evidence of acute cellular rejection. His clinical status deteriorated, he developed multiorgan system failure and died 12 days after his transplantation. No septic focus was identified. Both kidneys harvested from the same donor did not present any signs of delayed graft function after transplantation. Figure 1 A:  Serum Bilirubin levels (μmol/L) for patient 1 (solid line) and for patient 2 (dashed line). The arrowheads indicate the time points where calcifications were detected.  B:  International Normalization Ratio (INR) for patient 1 (solid line) and for patient 2 (dashed line). Arrowheads indicate the time points where calcifications were detected.  C:  Computed Tomography of patient 2. The arrow points an area in the right liver with the same density as the spinal column (arrowhead).  D:  Picture of the explant liver during retransplantation of the same patient (case 2). The arrow shows the abnormal area of the right liver correlating with the computed tomography findings.  E:  Light microscopy of epoxy embedded semi-section obtained from the liver biopsy from patient 1. The image shows moderate calcification (microcalcification) throughout the section. The arrow indicates a representative pattern of calcification.  F:  Light microscopy of epoxy embedded semi-section obtained from the tissues of the explant, following liver retransplantation of patient 2. The image shows the interface between calcified region (upper right) and non-calcified adjacent hepatic cells (lower left region).  G:  Light microscopy image showing a higher magnification of calcified region as shown in F. The bright and high contrast regions represent massive mineralization of hepatic cells of the explant, following retransplantation.  H:  Transmission electron microscopy images of ultrathin section obtained from the transitional zone between calcified and non-calcified tissue. Showing the mode of calcification and textural organization of hydroxyapatite crystal aggregates (dark contrast) within cytoplasmic region of the cell. Note alteration of the nucleus in the center. Case 2 A 55-year-old man of Greek origin underwent LT at our institution for ethanol and hepatitis B related cirrhosis, as well as a large hepatocellular carcinoma. The patient did not have a history suggestive of hyperparathyroidism or abnormal calcium metabolism, and upon listing his serum ionized calcium was 0.94 mmol/L. The donor was a previously healthy, 52 year-old male who suffered a closed head injury during a motor vehicle crash. The organ was procured by our transplant team. Prior to procurement the donor did not suffer any periods of hypoxia or hypotension. His liver function tests were normal prior to harvesting (AST 36 U/L, ALT 45 U/L). During procurement the liver was found to be well perfused, with no evidence of aberrant vascular anatomy and no evidence of trauma. Macroscopic examination of the liver did not show any evidence of steatosis. The recipient underwent a conventional LT without venovenous bypass and without intraoperative hypotension. The cold ischemic time was 10 hours while the warm ischemic time was 40 minutes. Following reperfusion, the graft appeared well perfused and again no biopsies were taken. Intraoperatively the patient received 500 mg of methylprednisolone, and postoperatively he was induced with Antithymocyte Globulin. During transplantation the recipient received 3 units of PRBCs and 6 units of FFP. In the early postoperative period the patient had to be explored once for retroperitoneal bleeding associated with hypotension (SBP< 90 mm Hg). At the time of LT, the patient had an INR of 2.7 and a serum total bilirubin of 104 μmol/L. Following LT, the patient developed severe primary graft dysfunction with rising serum bilirubin and INR (Figure  1A,1B ), while his serum ALT and AST peaked during the 2 nd  and 3 rd  postoperative day (4700 U/L and 6598 U/L respectively). At that time, ultrasonography demonstrated uniformly patent vessels (hepatic artery, portal and hepatic veins), while Computed Tomography (CT) showed areas in the right lobe of the liver isodense with the spinal column (Figure  1C ). The patient was listed for retransplantation. During the retransplant procedure the explant liver graft had a "bony" consistency in the involved right lobe. Cross-sections of the right lobe showed a "clay-like" parenchyma with clear evidence of calcification (Figure  1D ). Light (LM) and transmission electron microscopy (TEM) investigation of ultrathin sections obtained from specimens selected from biopsies at the interface of calcified and non-calcified tissues showed extensive intracellular calcification within the hepatic cells (Figure  1F ). High-resolution TEM (HRTEM) images and selected-area electron diffraction (SAED) combined with energy dispersive spectroscopy (EDS) analysis demonstrated the presence of hydroxyapatite (HA) as a solid phase in the calcified region. The adjacent non- or partially calcified hepatic cells displayed extensive nuclear condensation suggestive of significant apoptosis as well as severe vacuolization, suggestive of an extensive apoptotic and necrotic process (Figure  1G,1H ). The patient had a complicated postoperative course and finally died from ventricular fibrillation unresponsive to electrical cardioversion. Both kidneys harvested from the initial donor were transplanted without any evidence of delayed or primary graft dysfunction/non-function.
Case Presentations
Case 1 A 65-year-old man of Asian origin underwent LT at our institution on June 2002, for decompensated Hepatitis B related cirrhosis. The patient did not have a history of abnormal calcium metabolism, or hyperparathyroidism. Upon listing his serum ionized calcium was 1.07 mmol/L. The donor was a previously healthy, 42-year-old male, who suffered intracranial bleeding following a motor vehicle crash. He did not suffer any period of hypoxia but he was hypotensive (SBP = 90 mm Hg) prior to procurement. His serum liver function tests were normal prior to harvesting (AST 32 U/L, ALT 40 U/L). The organ was procured by our institution's transplant team. During procurement the liver was found to be well perfused with no focal injuries and no macroscopic evidence of steatosis. The recipient underwent an uncomplicated conventional LT without the use of a veno-venous bypass. There were no periods of hypoxia or severe hypotension during transplantation. The cold ischemic time of the graft was 8 hours while the warm ischemic time was 45 minutes. The graft reperfused well and no biopsies were taken. Intravenous methylprednisolone (500 mg) was administered intraoperatively, and postoperatively the patient received induction with Antithymocyte Globulin, which is the protocol followed at our institution. During transplantation the patient received a total of 4 units of packed red blood cells (PRBCs) and 6 units of fresh frozen plasma (FFP). At the time of LT, the international normalization ratio (INR) was 1.53, while serum total bilirubin was 103 μmol/L (Figure  1A,1B ). On postoperative day 2 the patient had a peak of his serum AST (3469 U/L) and at this point he had further biochemical evidence of primary graft dysfunction, with inability to normalize his INR (Figure  1B ), and with progressive elevation of his total serum bilirubin (Figure  1A ). Repeated ultrasonographic examination revealed a patent hepatic artery and portal vein, as well as patent hepatic veins. At this point, a liver biopsy demonstrated severe reperfusion injury with several apoptotic bodies, several dystrophic calcifications (Figure  1E ), and no evidence of acute cellular rejection. His clinical status deteriorated, he developed multiorgan system failure and died 12 days after his transplantation. No septic focus was identified. Both kidneys harvested from the same donor did not present any signs of delayed graft function after transplantation. Figure 1 A:  Serum Bilirubin levels (μmol/L) for patient 1 (solid line) and for patient 2 (dashed line). The arrowheads indicate the time points where calcifications were detected.  B:  International Normalization Ratio (INR) for patient 1 (solid line) and for patient 2 (dashed line). Arrowheads indicate the time points where calcifications were detected.  C:  Computed Tomography of patient 2. The arrow points an area in the right liver with the same density as the spinal column (arrowhead).  D:  Picture of the explant liver during retransplantation of the same patient (case 2). The arrow shows the abnormal area of the right liver correlating with the computed tomography findings.  E:  Light microscopy of epoxy embedded semi-section obtained from the liver biopsy from patient 1. The image shows moderate calcification (microcalcification) throughout the section. The arrow indicates a representative pattern of calcification.  F:  Light microscopy of epoxy embedded semi-section obtained from the tissues of the explant, following liver retransplantation of patient 2. The image shows the interface between calcified region (upper right) and non-calcified adjacent hepatic cells (lower left region).  G:  Light microscopy image showing a higher magnification of calcified region as shown in F. The bright and high contrast regions represent massive mineralization of hepatic cells of the explant, following retransplantation.  H:  Transmission electron microscopy images of ultrathin section obtained from the transitional zone between calcified and non-calcified tissue. Showing the mode of calcification and textural organization of hydroxyapatite crystal aggregates (dark contrast) within cytoplasmic region of the cell. Note alteration of the nucleus in the center.
Case 1
A 65-year-old man of Asian origin underwent LT at our institution on June 2002, for decompensated Hepatitis B related cirrhosis. The patient did not have a history of abnormal calcium metabolism, or hyperparathyroidism. Upon listing his serum ionized calcium was 1.07 mmol/L. The donor was a previously healthy, 42-year-old male, who suffered intracranial bleeding following a motor vehicle crash. He did not suffer any period of hypoxia but he was hypotensive (SBP = 90 mm Hg) prior to procurement. His serum liver function tests were normal prior to harvesting (AST 32 U/L, ALT 40 U/L). The organ was procured by our institution's transplant team. During procurement the liver was found to be well perfused with no focal injuries and no macroscopic evidence of steatosis.The recipient underwent an uncomplicated conventional LT without the use of a veno-venous bypass. There were no periods of hypoxia or severe hypotension during transplantation. The cold ischemic time of the graft was 8 hours while the warm ischemic time was 45 minutes. The graft reperfused well and no biopsies were taken. Intravenous methylprednisolone (500 mg) was administered intraoperatively, and postoperatively the patient received induction with Antithymocyte Globulin, which is the protocol followed at our institution. During transplantation the patient received a total of 4 units of packed red blood cells (PRBCs) and 6 units of fresh frozen plasma (FFP).At the time of LT, the international normalization ratio (INR) was 1.53, while serum total bilirubin was 103 μmol/L (Figure  1A,1B ). On postoperative day 2 the patient had a peak of his serum AST (3469 U/L) and at this point he had further biochemical evidence of primary graft dysfunction, with inability to normalize his INR (Figure  1B ), and with progressive elevation of his total serum bilirubin (Figure  1A ). Repeated ultrasonographic examination revealed a patent hepatic artery and portal vein, as well as patent hepatic veins. At this point, a liver biopsy demonstrated severe reperfusion injury with several apoptotic bodies, several dystrophic calcifications (Figure  1E ), and no evidence of acute cellular rejection. His clinical status deteriorated, he developed multiorgan system failure and died 12 days after his transplantation. No septic focus was identified. Both kidneys harvested from the same donor did not present any signs of delayed graft function after transplantation.Case 2 A 55-year-old man of Greek origin underwent LT at our institution for ethanol and hepatitis B related cirrhosis, as well as a large hepatocellular carcinoma. The patient did not have a history suggestive of hyperparathyroidism or abnormal calcium metabolism, and upon listing his serum ionized calcium was 0.94 mmol/L. The donor was a previously healthy, 52 year-old male who suffered a closed head injury during a motor vehicle crash. The organ was procured by our transplant team. Prior to procurement the donor did not suffer any periods of hypoxia or hypotension. His liver function tests were normal prior to harvesting (AST 36 U/L, ALT 45 U/L). During procurement the liver was found to be well perfused, with no evidence of aberrant vascular anatomy and no evidence of trauma. Macroscopic examination of the liver did not show any evidence of steatosis. The recipient underwent a conventional LT without venovenous bypass and without intraoperative hypotension. The cold ischemic time was 10 hours while the warm ischemic time was 40 minutes. Following reperfusion, the graft appeared well perfused and again no biopsies were taken. Intraoperatively the patient received 500 mg of methylprednisolone, and postoperatively he was induced with Antithymocyte Globulin. During transplantation the recipient received 3 units of PRBCs and 6 units of FFP. In the early postoperative period the patient had to be explored once for retroperitoneal bleeding associated with hypotension (SBP< 90 mm Hg). At the time of LT, the patient had an INR of 2.7 and a serum total bilirubin of 104 μmol/L. Following LT, the patient developed severe primary graft dysfunction with rising serum bilirubin and INR (Figure  1A,1B ), while his serum ALT and AST peaked during the 2 nd  and 3 rd  postoperative day (4700 U/L and 6598 U/L respectively). At that time, ultrasonography demonstrated uniformly patent vessels (hepatic artery, portal and hepatic veins), while Computed Tomography (CT) showed areas in the right lobe of the liver isodense with the spinal column (Figure  1C ). The patient was listed for retransplantation. During the retransplant procedure the explant liver graft had a "bony" consistency in the involved right lobe. Cross-sections of the right lobe showed a "clay-like" parenchyma with clear evidence of calcification (Figure  1D ). Light (LM) and transmission electron microscopy (TEM) investigation of ultrathin sections obtained from specimens selected from biopsies at the interface of calcified and non-calcified tissues showed extensive intracellular calcification within the hepatic cells (Figure  1F ). High-resolution TEM (HRTEM) images and selected-area electron diffraction (SAED) combined with energy dispersive spectroscopy (EDS) analysis demonstrated the presence of hydroxyapatite (HA) as a solid phase in the calcified region. The adjacent non- or partially calcified hepatic cells displayed extensive nuclear condensation suggestive of significant apoptosis as well as severe vacuolization, suggestive of an extensive apoptotic and necrotic process (Figure  1G,1H ). The patient had a complicated postoperative course and finally died from ventricular fibrillation unresponsive to electrical cardioversion. Both kidneys harvested from the initial donor were transplanted without any evidence of delayed or primary graft dysfunction/non-function.
Case 2
A 55-year-old man of Greek origin underwent LT at our institution for ethanol and hepatitis B related cirrhosis, as well as a large hepatocellular carcinoma. The patient did not have a history suggestive of hyperparathyroidism or abnormal calcium metabolism, and upon listing his serum ionized calcium was 0.94 mmol/L. The donor was a previously healthy, 52 year-old male who suffered a closed head injury during a motor vehicle crash. The organ was procured by our transplant team. Prior to procurement the donor did not suffer any periods of hypoxia or hypotension. His liver function tests were normal prior to harvesting (AST 36 U/L, ALT 45 U/L). During procurement the liver was found to be well perfused, with no evidence of aberrant vascular anatomy and no evidence of trauma. Macroscopic examination of the liver did not show any evidence of steatosis. The recipient underwent a conventional LT without venovenous bypass and without intraoperative hypotension. The cold ischemic time was 10 hours while the warm ischemic time was 40 minutes. Following reperfusion, the graft appeared well perfused and again no biopsies were taken. Intraoperatively the patient received 500 mg of methylprednisolone, and postoperatively he was induced with Antithymocyte Globulin. During transplantation the recipient received 3 units of PRBCs and 6 units of FFP. In the early postoperative period the patient had to be explored once for retroperitoneal bleeding associated with hypotension (SBP< 90 mm Hg).At the time of LT, the patient had an INR of 2.7 and a serum total bilirubin of 104 μmol/L. Following LT, the patient developed severe primary graft dysfunction with rising serum bilirubin and INR (Figure  1A,1B ), while his serum ALT and AST peaked during the 2 nd  and 3 rd  postoperative day (4700 U/L and 6598 U/L respectively). At that time, ultrasonography demonstrated uniformly patent vessels (hepatic artery, portal and hepatic veins), while Computed Tomography (CT) showed areas in the right lobe of the liver isodense with the spinal column (Figure  1C ). The patient was listed for retransplantation. During the retransplant procedure the explant liver graft had a "bony" consistency in the involved right lobe. Cross-sections of the right lobe showed a "clay-like" parenchyma with clear evidence of calcification (Figure  1D ). Light (LM) and transmission electron microscopy (TEM) investigation of ultrathin sections obtained from specimens selected from biopsies at the interface of calcified and non-calcified tissues showed extensive intracellular calcification within the hepatic cells (Figure  1F ). High-resolution TEM (HRTEM) images and selected-area electron diffraction (SAED) combined with energy dispersive spectroscopy (EDS) analysis demonstrated the presence of hydroxyapatite (HA) as a solid phase in the calcified region. The adjacent non- or partially calcified hepatic cells displayed extensive nuclear condensation suggestive of significant apoptosis as well as severe vacuolization, suggestive of an extensive apoptotic and necrotic process (Figure  1G,1H ).The patient had a complicated postoperative course and finally died from ventricular fibrillation unresponsive to electrical cardioversion. Both kidneys harvested from the initial donor were transplanted without any evidence of delayed or primary graft dysfunction/non-function.Conclusions Currently, more than 16,000 candidates are listed with the United Network for Organ Sharing awaiting liver transplantation. Nevertheless, only 4800 cadaveric liver transplants are performed annually in the United States. Due to this discordance between organ demand and supply, it is estimated that approximately 10% of patients in the waiting list will die before obtaining an organ. As a result, novel strategies to expand the donor pool have been explored. With the exception of live donor liver transplantation, the remaining strategies involve the use of older cadaveric grafts, allografts with mild steatosis or even donors with evidence of past hepatitis B or C infection [ 6 ]. This is why, one of the major obstacles to be tackled, is the development of clinically significant ischemia and reperfusion injury, which is even more important for "marginal" grafts. Every progress towards understanding the molecular events following not only cold storage but also cold and warm reperfusion of the graft could have a significant impact on the current transplantation practices. Indeed, recent data suggest that following I/R there is a balanced apoptosis and occasionally necrosis of hepatocytes translating into cell swelling, distension of various cellular organelles, clumping and random degradation of nuclear DNA, extensive plasma membrane endocytosis and autophagy [ 7 ]. Furthermore, when these events become predominant, they can lead, at least in animal models, to the development of calcifications as observed in livers of rabbits infected with rabbit haemorrhagic virus [ 2 ]. In the present case report, we have shown that these events can take place in human subjects following liver transplantation. To our knowledge these are the first reported cases in the literature of liver calcification following liver transplantation, presumably secondary to I/R injury. Not only did both patients have biochemical evidence of severe graft I/R injury, they also had biopsy proven I/R induced injury associated with the development of calcifications. Furthermore, both succumbed to the sequelae of this injury. Both recipients received grafts from donors with normal serum biochemistries and no evidence of hepatic trauma or steatosis. Both donors had no evidence of crystal deposition or storage disease, and although we did not perform any donor liver biopsies, the grafts appeared macroscopically normal and perfused well with UW solution. Corroborating to this remains the fact that all four kidneys (from both donors) were transplanted without any problems. Neither recipient had any evidence of calcium metabolism problems, since both had normal serum calcium upon listing. Both recipients had an anticipated intraoperative course without periods of hypotension and without massive transfusion requirements. Finally, both grafts did not demonstrate any vascular problems in the postoperative period by Ultrasonography or CT scan examination or any evidence of intrahepatic thrombosis in postmortem or explant examination. Ischemic stress has been previously reported to induce calcium accumulation at the cell level, either by impaired energy metabolism and/or plasmalemmal alterations. This elevated intracellular calcium concentration is responsible for cytoskeletal modifications, which alter cell shape, and for the activation of phospholipases, which results in perpetuation of membrane damage and finally, mitochondrial calcification [ 8 ]. Although, the crystal shape, composition and organization of HA in our samples are similar to those observed in bone and cartilage [ 9 ], as well as synthetic HA formed in serum [10], the intracellular precipitation of HA within hepatic cells is unique and has not been reported from other physiological and pathological tissues. The observation of calcified, vacuole-like structures in hepatic cells from these two livers could be suggestive of mitochondrial calcification. In addition, the extensive presence of phagocytic structures in the pre-calcified regions of these livers suggests an intense apoptotic/necrotic process undergone after I/R injury in these regions. Further investigation, however, is required to understand the mechanism(s) and the mode of calcification in the liver. In conclusion, we believe that the described phenomenon is underreported at least in the liver transplant literature. Furthermore, it appears that there is a correlation between the development of severe I/R injury leading to apoptosis and/or necrosis and calcifications detectable even by light microscopy. We think that the development of microcalcifications should be studied more extensively in the context of I/R injury following liver transplantation. Although, such a phenomenon appears to correlate with significant I/R injury, evident by biochemical data, it has the potential to be provide further information on the pathways of severe I/R injury post transplant.
Conclusions
Currently, more than 16,000 candidates are listed with the United Network for Organ Sharing awaiting liver transplantation. Nevertheless, only 4800 cadaveric liver transplants are performed annually in the United States. Due to this discordance between organ demand and supply, it is estimated that approximately 10% of patients in the waiting list will die before obtaining an organ. As a result, novel strategies to expand the donor pool have been explored. With the exception of live donor liver transplantation, the remaining strategies involve the use of older cadaveric grafts, allografts with mild steatosis or even donors with evidence of past hepatitis B or C infection [ 6 ]. This is why, one of the major obstacles to be tackled, is the development of clinically significant ischemia and reperfusion injury, which is even more important for "marginal" grafts. Every progress towards understanding the molecular events following not only cold storage but also cold and warm reperfusion of the graft could have a significant impact on the current transplantation practices. Indeed, recent data suggest that following I/R there is a balanced apoptosis and occasionally necrosis of hepatocytes translating into cell swelling, distension of various cellular organelles, clumping and random degradation of nuclear DNA, extensive plasma membrane endocytosis and autophagy [ 7 ]. Furthermore, when these events become predominant, they can lead, at least in animal models, to the development of calcifications as observed in livers of rabbits infected with rabbit haemorrhagic virus [ 2 ].In the present case report, we have shown that these events can take place in human subjects following liver transplantation. To our knowledge these are the first reported cases in the literature of liver calcification following liver transplantation, presumably secondary to I/R injury. Not only did both patients have biochemical evidence of severe graft I/R injury, they also had biopsy proven I/R induced injury associated with the development of calcifications. Furthermore, both succumbed to the sequelae of this injury. Both recipients received grafts from donors with normal serum biochemistries and no evidence of hepatic trauma or steatosis. Both donors had no evidence of crystal deposition or storage disease, and although we did not perform any donor liver biopsies, the grafts appeared macroscopically normal and perfused well with UW solution. Corroborating to this remains the fact that all four kidneys (from both donors) were transplanted without any problems. Neither recipient had any evidence of calcium metabolism problems, since both had normal serum calcium upon listing. Both recipients had an anticipated intraoperative course without periods of hypotension and without massive transfusion requirements. Finally, both grafts did not demonstrate any vascular problems in the postoperative period by Ultrasonography or CT scan examination or any evidence of intrahepatic thrombosis in postmortem or explant examination.Ischemic stress has been previously reported to induce calcium accumulation at the cell level, either by impaired energy metabolism and/or plasmalemmal alterations. This elevated intracellular calcium concentration is responsible for cytoskeletal modifications, which alter cell shape, and for the activation of phospholipases, which results in perpetuation of membrane damage and finally, mitochondrial calcification [ 8 ].Although, the crystal shape, composition and organization of HA in our samples are similar to those observed in bone and cartilage [ 9 ], as well as synthetic HA formed in serum [10], the intracellular precipitation of HA within hepatic cells is unique and has not been reported from other physiological and pathological tissues.The observation of calcified, vacuole-like structures in hepatic cells from these two livers could be suggestive of mitochondrial calcification. In addition, the extensive presence of phagocytic structures in the pre-calcified regions of these livers suggests an intense apoptotic/necrotic process undergone after I/R injury in these regions. Further investigation, however, is required to understand the mechanism(s) and the mode of calcification in the liver.In conclusion, we believe that the described phenomenon is underreported at least in the liver transplant literature. Furthermore, it appears that there is a correlation between the development of severe I/R injury leading to apoptosis and/or necrosis and calcifications detectable even by light microscopy. We think that the development of microcalcifications should be studied more extensively in the context of I/R injury following liver transplantation. Although, such a phenomenon appears to correlate with significant I/R injury, evident by biochemical data, it has the potential to be provide further information on the pathways of severe I/R injury post transplant.Competing Interests None declared.
Competing Interests
None declared.Authors Contributions GNT: conceived the study and wrote the manuscript MA: carried out the electron microscopy studies EC: participated in the design and analyzed the light microscopy results, critical review of the manuscript AE: analyzed light microscopy results AV: carried out electron microscopy studies PM: participated in the design of the study All authors read and approved the final manuscript.
Authors Contributions
GNT: conceived the study and wrote the manuscriptMA: carried out the electron microscopy studiesEC: participated in the design and analyzed the light microscopy results, critical review of the manuscriptAE: analyzed light microscopy resultsAV: carried out electron microscopy studiesPM: participated in the design of the studyAll authors read and approved the final manuscript.Abbreviations I/R:  Ischemia and Reperfusion LM:  Light Microscopy TEM:  Transmission Electron Microscopy HRTEM:  High-resolution Transmission Electron Microscopy EDS:  Energy Dispersive Spectroscopy HA:  Hydroxyapatite
Abbreviations
I/R:  Ischemia and ReperfusionI/R:LM:  Light MicroscopyLM:TEM:  Transmission Electron MicroscopyTEM:HRTEM:  High-resolution Transmission Electron MicroscopyHRTEM:EDS:  Energy Dispersive SpectroscopyEDS:HA:  HydroxyapatiteHA:Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here:


Background To evaluate the efficacy of diode laser photocoagulation in patients with central serous retinopathy (CSR) and to compare it with the effects of argon green laser.
Background
To evaluate the efficacy of diode laser photocoagulation in patients with central serous retinopathy (CSR) and to compare it with the effects of argon green laser.Methods Thirty patients with type 1 unilateral CSR were enrolled and evaluated on parameters like best corrected visual acuity (BCVA), direct and indirect ophthalmoscopy, amsler grid for recording scotoma and metamorphopsia, contrast sensitivity using Cambridge low contrast gratings and fluorescein angiography to determine the site of leakage. Patients were randomly assigned into 2 groups according to the statistical random table using sequence generation. In Group 1 (n = 15), diode laser (810 nm) photocoagulation was performed at the site of leakage while in Group 2 (n = 15), eyes were treated with argon green laser (514 nm) using the same laser parameters. Patients were followed up at 4, 8 and 12 weeks after laser.
Methods
Thirty patients with type 1 unilateral CSR were enrolled and evaluated on parameters like best corrected visual acuity (BCVA), direct and indirect ophthalmoscopy, amsler grid for recording scotoma and metamorphopsia, contrast sensitivity using Cambridge low contrast gratings and fluorescein angiography to determine the site of leakage.Patients were randomly assigned into 2 groups according to the statistical random table using sequence generation. In Group 1 (n = 15), diode laser (810 nm) photocoagulation was performed at the site of leakage while in Group 2 (n = 15), eyes were treated with argon green laser (514 nm) using the same laser parameters. Patients were followed up at 4, 8 and 12 weeks after laser.Results The mean BCVA in group 1 improved from a pre-laser decimal value of 0.29 ± 0.14 to 0.84 ± 0.23 at 4 weeks and 1.06 ± 0.09 at 12 weeks following laser. In group 2, the same improved from 0.32 ± 0.16 to 0.67 ± 0.18 at 4 weeks and 0.98 ± 0.14 at 12 weeks following laser. The improvement in BCVA was significantly better in group 1 (p < 0.0001) at 4 weeks. At 4 weeks following laser, all the patients in group1 were free of scotoma while 6 patients in group 2 had residual scotoma (p < 0.05). The mean contrast sensitivity in group 1 improved from pre-laser value of 98.4 ± 24.77 to 231.33 ± 48.97 at 4 weeks and 306.00 ± 46.57 at 12 weeks following laser. In group 2, the same improved from 130.66 ± 31.95 to 190.66 ± 23.44 at 4 weeks and 215.33 ± 23.25 at 12 weeks. On comparative evaluation, a significantly better (p < 0.001) improvement was noted in group 1.
Results
The mean BCVA in group 1 improved from a pre-laser decimal value of 0.29 ± 0.14 to 0.84 ± 0.23 at 4 weeks and 1.06 ± 0.09 at 12 weeks following laser. In group 2, the same improved from 0.32 ± 0.16 to 0.67 ± 0.18 at 4 weeks and 0.98 ± 0.14 at 12 weeks following laser. The improvement in BCVA was significantly better in group 1 (p < 0.0001) at 4 weeks. At 4 weeks following laser, all the patients in group1 were free of scotoma while 6 patients in group 2 had residual scotoma (p < 0.05). The mean contrast sensitivity in group 1 improved from pre-laser value of 98.4 ± 24.77 to 231.33 ± 48.97 at 4 weeks and 306.00 ± 46.57 at 12 weeks following laser. In group 2, the same improved from 130.66 ± 31.95 to 190.66 ± 23.44 at 4 weeks and 215.33 ± 23.25 at 12 weeks. On comparative evaluation, a significantly better (p < 0.001) improvement was noted in group 1.Conclusion Diode laser may be a better alternative to argon green laser whenever laser treatment becomes indicated in patients with central serous retinopathy in terms of faster visual rehabilitation and better contrast sensitivity. In addition, diode laser also has the well-recognized ergonomic and economic advantages.
Conclusion
Diode laser may be a better alternative to argon green laser whenever laser treatment becomes indicated in patients with central serous retinopathy in terms of faster visual rehabilitation and better contrast sensitivity. In addition, diode laser also has the well-recognized ergonomic and economic advantages.

Background Central serous retinopathy (CSR) is a retinal disorder affecting young adults, characterized clinically by a well-defined, translucent, circumscribed detachment of neurosensory retina at the posterior pole, usually involving the macula. The detachment results from accumulation of transparent fluid in the potential space between retinal pigment epithelial layer and the neurosensory retina. However, the source of this fluid and the exact etiopathogenesis of this disorder remain ill understood. Maumenee [ 1 ] described the concept of leakage site within the retinal pigment epithelial layer following fluorescein angiographic studies in patients with central serous retinopathy. Recent studies [ 2 - 4 ], using indocyanine green angiography, have indicated that the possible site of primary pathology in central serous retinopathy is the choroidal vessels and the involvement of the pigment epithelial layer is only secondary to it. Although, other treatment modalities have been suggested for central serous retinopathy, argon green (514 nm) laser photocoagulation has been the most widely accepted modality. The 810 nm-diode laser owing to deeper penetration should prove to be more efficacious in the treatment of central serous retinopathy given the current thinking that CSR may primarily be a choroidal disease. The present study was undertaken to test the efficacy of diode laser photocoagulation in central serous retinopathy in comparison to argon green laser. According to the best of our knowledge, this is the first clinical study in which a comparative evaluation between the above two groups has been done in central serous retinopathy.
Background
Central serous retinopathy (CSR) is a retinal disorder affecting young adults, characterized clinically by a well-defined, translucent, circumscribed detachment of neurosensory retina at the posterior pole, usually involving the macula. The detachment results from accumulation of transparent fluid in the potential space between retinal pigment epithelial layer and the neurosensory retina. However, the source of this fluid and the exact etiopathogenesis of this disorder remain ill understood. Maumenee [ 1 ] described the concept of leakage site within the retinal pigment epithelial layer following fluorescein angiographic studies in patients with central serous retinopathy. Recent studies [ 2 - 4 ], using indocyanine green angiography, have indicated that the possible site of primary pathology in central serous retinopathy is the choroidal vessels and the involvement of the pigment epithelial layer is only secondary to it.Although, other treatment modalities have been suggested for central serous retinopathy, argon green (514 nm) laser photocoagulation has been the most widely accepted modality. The 810 nm-diode laser owing to deeper penetration should prove to be more efficacious in the treatment of central serous retinopathy given the current thinking that CSR may primarily be a choroidal disease. The present study was undertaken to test the efficacy of diode laser photocoagulation in central serous retinopathy in comparison to argon green laser. According to the best of our knowledge, this is the first clinical study in which a comparative evaluation between the above two groups has been done in central serous retinopathy.Methods Thirty patients of unilateral central serous retinopathy presenting to the retina services at the Rajendra Prasad Centre for Ophthalmic Sciences were selected for the study. They were randomly assigned into 2 groups according to the statistical random table using sequence generation. As it was a pilot study we decided only the convenient sample size i.e. 15 in each group. The allocation of patients into 2 groups was done by a person who was not involved in the study and the sequence was concealed until interventions were assigned to prevent bias. The patient selection was based on the following inclusion criteria: age less than 50 years, type 1 CSR with a single leak on fluorescein angiography that was at least 300 microns away from fovea, presence of an indication for laser treatment (recurrence, occupational, history of poor visual outcome in fellow eye) and no history of any treatment in the past. Patients with multiple leak CSR, type 2 or type 3 CSR or leak at papillomacular bundle or leak within 300 μ from the foveal centre were not included in the study. Patients with other ocular pathology such as choroidal neovascularization, choroidal inflammatory or neoplastic disorder or a congenital optic nerve pit were also excluded from the study. Informed consent was obtained from all the patients included in the study before initiating treatment and an approval of the institute body ethics committee was obtained to undertake the study. In all the patients the following parameters were evaluated before laser treatment and also during the follow-up period: best corrected visual acuity (BCVA) on Snellen's acuity chart, fundus examination by direct and indirect ophthalmoscopy, amsler grid for determining the degree of scotoma and / or metamorphopsia, contrast sensitivity using Cambridge low contrast gratings and fluorescein angiography (Canon CF-60 UD camera using 400ASA, 35-mm black and white film). Technique of scoring scotoma and metamorphopsia on the amsler grid is described below: The amsler grid consists of a 10-cm square divided into smaller 5 mm squares. When it is viewed at one-third of a meter, each small square subtends an angle of 1°. The patient was asked to look directly at the central spot with the uncovered eye and to mark out any black spot, distortion of lines, or blurred area on the grid. The number of small squares within the area delineated by the patient was counted and scotoma and metamorphopsia score was graded numerically as shown in table  1  and  2 . Patients included in the study were randomly assigned to one of the following two groups. In group 1 (n = 15), the site of leakage was treated by diode laser (810 nm). Laser was performed with Volk area centralis contact lens (Keeler Ltd, Clewer Hill Road, Windsor, Berks, SL4 4AA) and the parameters used were spot size of 100 microns, duration of 100 to 200 milliseconds and power just enough to produce minimal greying. In group 2 (n = 15), patients were treated with argon green laser (514 nm) following the same laser parameters. Patients were followed up at intervals of 4, 8 and 12 weeks after laser treatment and the results were analyzed, which included a comparative analysis of the outcome between the two groups. Descriptive statistics i.e. mean and standard deviation was calculated for each variable at each follow-up for the 2 groups. To see the significant difference between the 2 groups at each point we applied "student 't' test" (unpaired). To see the trend within the group we applied "two way analysis of variance (ANOVA)". 'P' value of 0.05 was considered as statistically significant. BMDP 7.0 statistical package was used for the statistical analysis.
Methods
Thirty patients of unilateral central serous retinopathy presenting to the retina services at the Rajendra Prasad Centre for Ophthalmic Sciences were selected for the study. They were randomly assigned into 2 groups according to the statistical random table using sequence generation. As it was a pilot study we decided only the convenient sample size i.e. 15 in each group. The allocation of patients into 2 groups was done by a person who was not involved in the study and the sequence was concealed until interventions were assigned to prevent bias. The patient selection was based on the following inclusion criteria: age less than 50 years, type 1 CSR with a single leak on fluorescein angiography that was at least 300 microns away from fovea, presence of an indication for laser treatment (recurrence, occupational, history of poor visual outcome in fellow eye) and no history of any treatment in the past. Patients with multiple leak CSR, type 2 or type 3 CSR or leak at papillomacular bundle or leak within 300 μ from the foveal centre were not included in the study. Patients with other ocular pathology such as choroidal neovascularization, choroidal inflammatory or neoplastic disorder or a congenital optic nerve pit were also excluded from the study.Informed consent was obtained from all the patients included in the study before initiating treatment and an approval of the institute body ethics committee was obtained to undertake the study. In all the patients the following parameters were evaluated before laser treatment and also during the follow-up period: best corrected visual acuity (BCVA) on Snellen's acuity chart, fundus examination by direct and indirect ophthalmoscopy, amsler grid for determining the degree of scotoma and / or metamorphopsia, contrast sensitivity using Cambridge low contrast gratings and fluorescein angiography (Canon CF-60 UD camera using 400ASA, 35-mm black and white film).Technique of scoring scotoma and metamorphopsia on the amsler grid is described below:The amsler grid consists of a 10-cm square divided into smaller 5 mm squares. When it is viewed at one-third of a meter, each small square subtends an angle of 1°. The patient was asked to look directly at the central spot with the uncovered eye and to mark out any black spot, distortion of lines, or blurred area on the grid. The number of small squares within the area delineated by the patient was counted and scotoma and metamorphopsia score was graded numerically as shown in table  1  and  2 .Patients included in the study were randomly assigned to one of the following two groups. In group 1 (n = 15), the site of leakage was treated by diode laser (810 nm). Laser was performed with Volk area centralis contact lens (Keeler Ltd, Clewer Hill Road, Windsor, Berks, SL4 4AA) and the parameters used were spot size of 100 microns, duration of 100 to 200 milliseconds and power just enough to produce minimal greying. In group 2 (n = 15), patients were treated with argon green laser (514 nm) following the same laser parameters. Patients were followed up at intervals of 4, 8 and 12 weeks after laser treatment and the results were analyzed, which included a comparative analysis of the outcome between the two groups. Descriptive statistics i.e. mean and standard deviation was calculated for each variable at each follow-up for the 2 groups. To see the significant difference between the 2 groups at each point we applied "student 't' test" (unpaired). To see the trend within the group we applied "two way analysis of variance (ANOVA)". 'P' value of 0.05 was considered as statistically significant. BMDP 7.0 statistical package was used for the statistical analysis.Results The average age of the patients in group 1 was 34.06 ± 2.54 years (range: 30–40 years) and in group 2, it was 34.66 ± 3.23 years (range: 30–42 years). Twelve cases (80%) in group 1 and 13 patients (86.66%) in group 2 were males. All the patients presented to us within eight days (range: 3–8 days) of onset of visual symptoms. The average number of laser spots applied to the leakage site in group 1 was 3.13 ± 0.71 (range: 2–4) and in group 2 it was 3.26 ± 0.77 (range: 2–4). All the patients came regularly for the follow-up as per the protocol during entire period of study. The study was performed during the period between January 1998 and June 2000. The mean best-corrected visual acuity (BCVA) in decimal at the time of presentation in patients assigned to group 1 was 0.29 ± 0.14. Best corrected visual acuity four weeks after diode laser treatment had improved to 0.84 ± 0.23. The BCVA further improved to 0.97 ± 0.17 and 1.06 ± 0.09 at 8 weeks and 12 weeks respectively. In group 2, the mean BCVA at the time of presentation was 0.32 ± 0.16 and it improved to 0.67 ± 0.18 at 4 weeks follow-up after argon green laser photocoagulation. Further improvement in visual acuity to 0.91 ± 0.19 and 0.98 ± 0.14 at 8 weeks and 12 weeks follow up respectively was observed. Statistical analysis of data pertaining to improvement of BCVA in the two groups by student  't'  test, showed a statistically significant difference at 4 weeks with patients treated by diode laser (group 1) having a better improvement (p < 0.001) (Figure  1 ). This difference decreased and was not found to be statistically significant during later follow-up at 8 and 12 weeks. All the patients in both the groups had central scotoma at the time of presentation. At the first follow up at 4 weeks following laser treatment, all the patients in group 1 (n = 15) were free of scotoma while 6 patients in group 2 (n = 15) had residual scotoma of grade 1 (involving 1–50 small squares on the amsler grid). On comparative evaluation, this difference was found to be statistically significant (p < 0.05). At 8 weeks and 12 weeks follow up, none of the patients had residual scotoma; however, 3 patients in group 2 developed a paracentral scotoma corresponding to the site of laser scar. Four patients in group 1 and five in group 2 had complaints of metamorphopsia at the time of presentation. At the first follow up at 4 weeks, 2 cases in group 1 and 4 cases in group 2 were still having grade 1 metamorphopsia. On comparative evaluation, this difference in the two groups was not found to be significant statistically. At 8 weeks and 12 weeks follow up, all patients in both the groups were free of metamorphopsia. Contrast sensitivity was evaluated using the Cambridge low contrast gratings. The reference normal for our patients as observed in earlier studies at our centre has been 340 (range 310–370) (unpublished data). The mean absolute value of contrast sensitivity at the time of presentation in group 1 was 98.4 ± 24.77. This improved to 231.33 ± 48.97 at the first follow up at 4 weeks following diode laser photocoagulation. It further improved to 286.00 ± 48.52 at 8 weeks follow up and 306.00 ± 46.57 at the third follow up at 12 weeks. In group 2, the mean value of contrast sensitivity at the time of presentation was 130.66 ± 31.95, which improved to 190.66 ± 23.44 at 4 weeks following argon green laser treatment. This value further improved to 210.00 ± 27.25 at 8 weeks and 215.33 ± 23.25 at 12 weeks follow up. Comparative evaluation of the improvement in contrast sensitivity by student  't'  test between the two groups showed that there was an initial rapid improvement in contrast sensitivity in both the groups but group I patients had a significantly greater improvement than group 2 patients at all the follow up visits (p < 0.001) (Figure  2 ). In both the groups, the commonest site of fluorescein leakage was the area superonasal to fovea. Fluorescein angiography repeated 4 weeks later showed that in both the groups, all the patients had complete resolution of the leakage site. No recurrence was reported till completion of follow up at 12 weeks.
Results
The average age of the patients in group 1 was 34.06 ± 2.54 years (range: 30–40 years) and in group 2, it was 34.66 ± 3.23 years (range: 30–42 years). Twelve cases (80%) in group 1 and 13 patients (86.66%) in group 2 were males. All the patients presented to us within eight days (range: 3–8 days) of onset of visual symptoms. The average number of laser spots applied to the leakage site in group 1 was 3.13 ± 0.71 (range: 2–4) and in group 2 it was 3.26 ± 0.77 (range: 2–4). All the patients came regularly for the follow-up as per the protocol during entire period of study. The study was performed during the period between January 1998 and June 2000.The mean best-corrected visual acuity (BCVA) in decimal at the time of presentation in patients assigned to group 1 was 0.29 ± 0.14. Best corrected visual acuity four weeks after diode laser treatment had improved to 0.84 ± 0.23. The BCVA further improved to 0.97 ± 0.17 and 1.06 ± 0.09 at 8 weeks and 12 weeks respectively. In group 2, the mean BCVA at the time of presentation was 0.32 ± 0.16 and it improved to 0.67 ± 0.18 at 4 weeks follow-up after argon green laser photocoagulation. Further improvement in visual acuity to 0.91 ± 0.19 and 0.98 ± 0.14 at 8 weeks and 12 weeks follow up respectively was observed. Statistical analysis of data pertaining to improvement of BCVA in the two groups by student  't'  test, showed a statistically significant difference at 4 weeks with patients treated by diode laser (group 1) having a better improvement (p < 0.001) (Figure  1 ). This difference decreased and was not found to be statistically significant during later follow-up at 8 and 12 weeks.'t'All the patients in both the groups had central scotoma at the time of presentation. At the first follow up at 4 weeks following laser treatment, all the patients in group 1 (n = 15) were free of scotoma while 6 patients in group 2 (n = 15) had residual scotoma of grade 1 (involving 1–50 small squares on the amsler grid). On comparative evaluation, this difference was found to be statistically significant (p < 0.05). At 8 weeks and 12 weeks follow up, none of the patients had residual scotoma; however, 3 patients in group 2 developed a paracentral scotoma corresponding to the site of laser scar.Four patients in group 1 and five in group 2 had complaints of metamorphopsia at the time of presentation. At the first follow up at 4 weeks, 2 cases in group 1 and 4 cases in group 2 were still having grade 1 metamorphopsia. On comparative evaluation, this difference in the two groups was not found to be significant statistically. At 8 weeks and 12 weeks follow up, all patients in both the groups were free of metamorphopsia.Contrast sensitivity was evaluated using the Cambridge low contrast gratings. The reference normal for our patients as observed in earlier studies at our centre has been 340 (range 310–370) (unpublished data). The mean absolute value of contrast sensitivity at the time of presentation in group 1 was 98.4 ± 24.77. This improved to 231.33 ± 48.97 at the first follow up at 4 weeks following diode laser photocoagulation. It further improved to 286.00 ± 48.52 at 8 weeks follow up and 306.00 ± 46.57 at the third follow up at 12 weeks. In group 2, the mean value of contrast sensitivity at the time of presentation was 130.66 ± 31.95, which improved to 190.66 ± 23.44 at 4 weeks following argon green laser treatment. This value further improved to 210.00 ± 27.25 at 8 weeks and 215.33 ± 23.25 at 12 weeks follow up. Comparative evaluation of the improvement in contrast sensitivity by student  't'  test between the two groups showed that there was an initial rapid improvement in contrast sensitivity in both the groups but group I patients had a significantly greater improvement than group 2 patients at all the follow up visits (p < 0.001) (Figure  2 ).'t'In both the groups, the commonest site of fluorescein leakage was the area superonasal to fovea. Fluorescein angiography repeated 4 weeks later showed that in both the groups, all the patients had complete resolution of the leakage site. No recurrence was reported till completion of follow up at 12 weeks.Discussion Visual prognosis in central serous retinopathy in most cases is good as they resolve spontaneously. Klein M et al. [ 5 ] studied and followed up 34 eyes of CSR without giving any treatment and found that final visual acuity was 20/30 or better. The average time for the fluid to resolve was between 3 months to 6 months and there was a recurrence rate of 45%. Sequelae such as macular degeneration and chronic retinal pigment epithelial decompensation are rare after the first episode. However, in a small group of patients visual prognosis has been reported to be less favorable [ 6 , 7 ]. Encouraging results of laser photocoagulation has been found in various studies which conclude that laser photocoagulation could effectively decrease the time for visual recovery in patients with central serous retinopathy [ 8 - 10 ]. However, the ultimate visual acuity despite laser photocoagulation remained no different from patients in whom no treatment was done and that recurrences could not be prevented by laser photocoagulation [ 10 ]. Results of treatment of the leakage site in patients with central serous retinopathy with ruby [ 9 ], krypton [ 11 , 12 ] and argon lasers [ 9 , 10 , 13 , 14 ] and Xenon arc [ 15 ] have been reported in the literature. Although, Slusher [ 11 ] suggested that the wavelength emitted by krypton laser might be more tissue-selective, argon green laser photocoagulation has been the most widely accepted treatment modality in patients with central serous retinopathy [ 9 , 10 , 12 - 14 ]. Studies performed with indocyanine green angiography in cases of central serous retinopathy have reported that hyperpermeability of choroidal vessels could be the primary pathology in CSR and retinal pigment epithelial abnormality is only secondary to it [ 2 - 4 ]. Brancato R et al [ 16 ] performed transpupillary chorioretinal photocoagulation in rabbit eyes using a diode laser prototype and a commercially available argon laser and found that they were similar on ophthalmoscopy. However, histopathological examination performed twenty-four hours after the treatment showed that argon irradiation resulted in damage to both the inner and outer retinal layers while the diode laser radiation produced damage to outer retina and choroid. If this were true, energy delivered by argon laser (514 nm) seems inadequate in being able to reach the deeper choroidal layers and treat the primary site of pathology. This inherent limitation of argon laser in view of recent ICG reports on the pathogenesis of central serous retinopathy could also explain the less than ideal outcome in the earlier studies using this laser in the treatment of patients with central serous retinopathy. The 810 nm-diode laser emission is in the near infrared (810 nm) region and this wavelength is able to reach the deeper choroidal layers [ 16 - 18 ]. Hence, we felt that the 810 nm-diode laser may theoretically be more efficacious in the laser treatment of central serous retinopathy. Our observations showed that following diode laser treatment in patients with central serous retinopathy, the recovery of visual acuity was more rapid in comparison to the argon laser treatment group. Further, all the patients treated with diode laser showed complete resolution of scotoma at the first follow up at 4 weeks and none of them developed a scotoma corresponding to the site of laser treatment. In contrast, in patients subjected to argon green laser treatment, there was a significant residual scotoma at 4 weeks and 20% of the patients developed scotoma corresponding to the laser scar. Following laser treatment, contrast sensitivity in both the groups improved rapidly in the first four weeks. However, there was significantly greater improvement in contrast sensitivity in eyes subjected to diode laser. This observation is indicative that diode laser is more beneficial with regard to conservation of contrast sensitivity following laser treatment in patients with central serous retinopathy. Development of subretinal neovascularization following laser treatment for central serous retinopathy has been described earlier in literature [ 19 ]. However, in our study none of the patients in any group developed this complication. The present study suggests that diode laser photocoagulation brings about a faster quantitative and qualitative visual recovery in comparison to argon green laser in eyes with central serous retinopathy. The main aim of performing laser photocoagulation in eyes with CSR is to hasten resolution and this is better fulfilled by diode laser. Further, as argon laser can hit up to the outer retinal layers, it can seal the site of leaking retinal pigment epithelium, but it cannot reduce the fluid overload over the pigment epithelial layer caused by hyperpermeability of choriocapillaries, which can be the cause of recurrence of CSR. As diode laser hits the choroid, which is the exact site of pathology, it should reduce the fluid overload on the retinal pigment epithelium.
Discussion
Visual prognosis in central serous retinopathy in most cases is good as they resolve spontaneously. Klein M et al. [ 5 ] studied and followed up 34 eyes of CSR without giving any treatment and found that final visual acuity was 20/30 or better. The average time for the fluid to resolve was between 3 months to 6 months and there was a recurrence rate of 45%. Sequelae such as macular degeneration and chronic retinal pigment epithelial decompensation are rare after the first episode. However, in a small group of patients visual prognosis has been reported to be less favorable [ 6 , 7 ].Encouraging results of laser photocoagulation has been found in various studies which conclude that laser photocoagulation could effectively decrease the time for visual recovery in patients with central serous retinopathy [ 8 - 10 ]. However, the ultimate visual acuity despite laser photocoagulation remained no different from patients in whom no treatment was done and that recurrences could not be prevented by laser photocoagulation [ 10 ].Results of treatment of the leakage site in patients with central serous retinopathy with ruby [ 9 ], krypton [ 11 , 12 ] and argon lasers [ 9 , 10 , 13 , 14 ] and Xenon arc [ 15 ] have been reported in the literature. Although, Slusher [ 11 ] suggested that the wavelength emitted by krypton laser might be more tissue-selective, argon green laser photocoagulation has been the most widely accepted treatment modality in patients with central serous retinopathy [ 9 , 10 , 12 - 14 ].Studies performed with indocyanine green angiography in cases of central serous retinopathy have reported that hyperpermeability of choroidal vessels could be the primary pathology in CSR and retinal pigment epithelial abnormality is only secondary to it [ 2 - 4 ].Brancato R et al [ 16 ] performed transpupillary chorioretinal photocoagulation in rabbit eyes using a diode laser prototype and a commercially available argon laser and found that they were similar on ophthalmoscopy. However, histopathological examination performed twenty-four hours after the treatment showed that argon irradiation resulted in damage to both the inner and outer retinal layers while the diode laser radiation produced damage to outer retina and choroid. If this were true, energy delivered by argon laser (514 nm) seems inadequate in being able to reach the deeper choroidal layers and treat the primary site of pathology. This inherent limitation of argon laser in view of recent ICG reports on the pathogenesis of central serous retinopathy could also explain the less than ideal outcome in the earlier studies using this laser in the treatment of patients with central serous retinopathy. The 810 nm-diode laser emission is in the near infrared (810 nm) region and this wavelength is able to reach the deeper choroidal layers [ 16 - 18 ]. Hence, we felt that the 810 nm-diode laser may theoretically be more efficacious in the laser treatment of central serous retinopathy.Our observations showed that following diode laser treatment in patients with central serous retinopathy, the recovery of visual acuity was more rapid in comparison to the argon laser treatment group. Further, all the patients treated with diode laser showed complete resolution of scotoma at the first follow up at 4 weeks and none of them developed a scotoma corresponding to the site of laser treatment. In contrast, in patients subjected to argon green laser treatment, there was a significant residual scotoma at 4 weeks and 20% of the patients developed scotoma corresponding to the laser scar.Following laser treatment, contrast sensitivity in both the groups improved rapidly in the first four weeks. However, there was significantly greater improvement in contrast sensitivity in eyes subjected to diode laser. This observation is indicative that diode laser is more beneficial with regard to conservation of contrast sensitivity following laser treatment in patients with central serous retinopathy.Development of subretinal neovascularization following laser treatment for central serous retinopathy has been described earlier in literature [ 19 ]. However, in our study none of the patients in any group developed this complication.The present study suggests that diode laser photocoagulation brings about a faster quantitative and qualitative visual recovery in comparison to argon green laser in eyes with central serous retinopathy. The main aim of performing laser photocoagulation in eyes with CSR is to hasten resolution and this is better fulfilled by diode laser. Further, as argon laser can hit up to the outer retinal layers, it can seal the site of leaking retinal pigment epithelium, but it cannot reduce the fluid overload over the pigment epithelial layer caused by hyperpermeability of choriocapillaries, which can be the cause of recurrence of CSR. As diode laser hits the choroid, which is the exact site of pathology, it should reduce the fluid overload on the retinal pigment epithelium.Conclusions In the present study, our observations are suggestive that diode laser may be a better alternative to argon laser whenever laser treatment is indicated in patients with central serous retinopathy. In addition, diode laser also has the well-recognized economic and ergonomic advantages of being less costly, more efficient, portable and easy to maintain [ 20 , 21 ], which is also a matter of concern particularly in third world countries.
Conclusions
In the present study, our observations are suggestive that diode laser may be a better alternative to argon laser whenever laser treatment is indicated in patients with central serous retinopathy. In addition, diode laser also has the well-recognized economic and ergonomic advantages of being less costly, more efficient, portable and easy to maintain [ 20 , 21 ], which is also a matter of concern particularly in third world countries.Declaration of competing interest The author(s) declare that they have no competing interests.
Declaration of competing interest
The author(s) declare that they have no competing interests.Authors' contributions LV designed the study and performed laser. RS performed the data collection and wrote the manuscript. PV followed up the patients. HKT performed the angiography. All authors read and approved the final manuscript. Presented in part in the Annual Meeting of the American Academy of Ophthalmology, 1997 in San Francisco, California.
Authors' contributions
LV designed the study and performed laser. RS performed the data collection and wrote the manuscript. PV followed up the patients. HKT performed the angiography.All authors read and approved the final manuscript.Presented in part in the Annual Meeting of the American Academy of Ophthalmology, 1997 in San Francisco, California.Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here: