Treating an unstable pelvic arterial bleed in angiography is not without risks. Angiography is often performed in remote locations in the hospital. The unstable patient would require transport to the angiography suite and then potentially to the operating rooms. An anesthesia team is often requested to accompany the patient in the angiography suite to perform continued monitoring, airway management, and resuscitation. The anesthesia equipment and monitors used in the remote location should meet the same standards as those used in the operating room. The Anesthesiologist must also have a reliable way to

Most reports suggest that the optimal time for stabilization of a pelvic fracture is within one week of the injury. (Miller, 2009) Recent studies have shown that there are benefits associated with an early (within 24 hours of injury) stabilization or reduction and definitive fixation of an unstable pelvic fracture. These benefits include: control of bleeding, assist with resuscitation, pain relief, ability to mobilize the patient, ease of reduction, improved reduction quality, elimination of traction, elimination of recumbency, reduced risk of pulmonary, septic, and thromboembolic complications, less organ failure, reduced morbidity and mortality, decreased length of stay in the intensive care unit, and shorter

A hip dislocation is a medical emergency, often resulting from a high impact trauma. Failure to quickly diagnose and treat this injury can result in avascular necrosis of the femoral head and significant neurologic injury. A hip dislocation usually requires a very deep level of anesthesia to facilitate a successful reduction. While occasionally the hip dislocation can be reduced in a spontaneously breathing patient with a combination of midazolam, fentanyl, ketamine, and/or propofol, often they require a nondepolarizing muscle relaxant. If a trauma patient requires complete relaxation with a nondepolaring muscle relaxant, then the procedure should be performed in the operating room with full ASA monitors and an endotracheal tube secondary to the trauma patients increased risk of aspiration. Patients that present to the hospital and are inebriated, uncooperative, confused, hemodynamically unstable, or have pulmonary dysfunction, should also be intubated

Hip fractures are most common in the elderly. They are associated with a significantly high morbidity and one-year mortality (30%). These patients often present in considerable pain,

resuscitation. (Miller, 2009)

communicate for help if needed. (Smith, 2008)

overall hospital stay. (Vallier, 2010)

prior to a hip reduction. (Miller, 2009)

**6.4 Hip fracture (femoral neck fracture)**

**6.3 Hip dislocation**

high stress states, and possibly exhibiting symptoms of myocardial ischemia. Early surgery (<12 hours) has resulted in: lower pain scores, decreased length of hospital stay, and reduced peri-operative complications. Compared with delayed surgery, there is no association with increased survival. Early surgery should be the goal for stable patients, combined with early mobilization, rehabilitation, and aggressive medical care. (Miller, 2009)

Hip fractures can result in significant blood loss. The patients are often dehydrated and anemic. A normal intravascular blood volume should be restored prior to anesthesia and surgery. Placement of a central venous catheter and arterial line can guide the fluid management and help prevent congestive heart failure from over-resuscitation. (Miller, 2009)

#### **Peri-operative Complications of Hip Fractures**

Table 27. Peri-operative complications of hip fractures (Miller, 2009)

Patients undergoing a hip fracture surgery have the highest risk of death from a pulmonary embolism. A spinal anesthetic is recommended for the actual surgery, but an epidural is rarely indicated for post-operative pain management secondary to the aggressive postoperative anticoagulation that will be initiated. (Miller, 2009)

#### **6.5 Open fracture**

Open fractures are surgical emergencies. The infection rate increases after a delay of 6-8 hours. Debridement and irrigation in the operating room plus provisional or definitive fixation of the fracture should happen as soon as it is safely possible. Open fractures require cleaning, pulse lavage, and debridement as soon as possible to minimize the risk of infectious complications. If the patient is not stable enough to go to the operating room, this should be done at the bedside. (Smith, 2008)

#### **6.6 Traumatic amputation**

A traumatic amputation requires immediate treatment with pressure, control of the bleeding, early intravenous antibiotics, and tetanus prophylaxis. Emergent surgery is often necessary to control the bleeding and perform surgical debridement. (Smith, 2008)

#### **6.7 Vascular injury**

"Injury to the major arterial flow of a limb is a surgical emergency." It is most common after a penetrating trauma, but can be seen with blunt injuries. Traumatic knee dislocations are the most common etiology for a vascular injury in a blunt trauma. A major arterial injury should be suspected in a patient that presents with pallor, coolness, and decreased pulses of the extremity. The patient may also present with an expanding hematoma or massive bleeding. Blood and fluid replacement by the anesthesia team is critical for the patient's survival. (Smith, 2008)

Anesthesia for Orthopedic Trauma 209

Patients with orthopedic injury and poly-trauma including orthopedic injury may present with a significant blood loss. Massive transfusion protocols (MTP) have been designed and implemented in many large urban trauma centers to provide a rapidly bleeding patient with automatic regular shipments of blood products to facilitate fluid resuscitation during an emergency surgery. The goal of the design ratio of packed red blood cells, plasma, and platelets built into the protocol are based on the need for volume support, oxygen delivery,

When should the MTP be initiated? This is often a judgment call made by the Surgeon or Anesthesiologist. It can be guided by these criteria: 5-unit blood loss in one hour, or 10-unit blood loss anticipated for the entire case or within 12-24 hours of observation, or hypovolemic hypotension uncorrected by crystalloid and/or packed red blood cell

During the MTP, a cooler containing blood products is brought one at a time to the operating room as each is finished or until the MTP is terminated. The MTP will vary from

3 5 2 10 1

Hypothermia is defined as a core temperature less than 36 °C. Hypothermia is usually caused by prolonged exposures to cold temperatures. Many trauma patients present to the operating room very cold secondary to prolonged exposure to the elements after the initial trauma. Mild hypothermia, about 1-2 °C below normal triples the incidence of morbid cardiac outcomes, triples the incidence of wound infections, directly impairs immune function, prolongs hospitalization by up to 20%, significantly increases surgical blood loss,

Trauma patients may enter the operating room already cold, but even if they are normothermic, the patients' core temperatures usually decrease by 0.5-1.5 °C in the first 30 minutes after the induction of anesthesia secondary to the internal redistribution of heat. Mechanisms for heat loss include radiation loss, convection loss, conduction loss, and

**5 Pack of**

**Platelets Cryoprecipitate Recombinant**

**factor VIIa**

and coagulation support required at each stage of the resuscitation. (Smith, 2008)

resuscitation during an ongoing hemorrhage. (Smith, 2008)

hospital to hospital. See below for a sample MTP. (Smith, 2008)

negative) 2 (AB)

2 5 2 1

4 5 2 1

Table 29. Sample massive transfusion protocol (MTP) (Smith, 2008)

and can lead to cold-induced platelet dysfunction. (Lovich-Sapola, 2010)

**Thawed Plasma**

**7.2 Significant blood loss**

**Cooler Packed Red**

1a (No type and screen available.)

1b (Type and

**7.3 Hypothermia**

**Blood Cells**

5 (O

Crossed) 5 2

evaporative loss. (Lovich-Sapola, 2010)

### **7. Intra-operative complications**

#### **7.1 Hypotension**

Hypotension can occur during any case in the operating room. In a trauma situation, it is especially important to consider a wide differential so that the true cause of the hypotension is not missed. Hypotension may be caused by hypovolemia: undetected or underestimated blood loss, insensible losses, gastrointestinal loss, renal loss, excessive venodilation, and redistribution of fluid to an extravascular space. All forms of shock including obstructive, cardiogenic, and vasodilated shock should be ruled out. (Smith, 2008)

Table 28. Types of shock (Smith, 2008)

Resuscitation from shock refers to the restoration of normal physiology after injury. Resuscitation after a hemorrhagic shock requires the restoration of a normal circulating blood volume, normal vascular tone, and normal tissue perfusion. (Miller, 2009)

Early resuscitation occurs while active bleeding is still ongoing. The goals in early resuscitation are to maintain the systolic blood pressure at 80 to 100 mmHg, maintain the hematocrit at 25-30%, maintain the coagulation panel and ionized calcium level within a normal range, keep the platelet count > 50,000, maintain the core temperature at > 35 °C, and prevent worsening acidosis. Late resuscitation is defined as after the bleeding is controlled and has separate goals that include maintaining the systolic blood pressure > 100 mmHg, maintaining a normal hematocrit, normalizing the coagulation status, electrolytes, temperature, and urine output, and maximizing the cardiac output. (Miller, 2009)

#### **7.2 Significant blood loss**

208 Orthopedic Surgery

Hypotension can occur during any case in the operating room. In a trauma situation, it is especially important to consider a wide differential so that the true cause of the hypotension is not missed. Hypotension may be caused by hypovolemia: undetected or underestimated blood loss, insensible losses, gastrointestinal loss, renal loss, excessive venodilation, and redistribution of fluid to an extravascular space. All forms of shock including obstructive,

**Type of shock Differential Diagnoses**

 Pericardial tamponade Massive pleural effusion

 Abdominal compartment syndrome Arterial or venous occlusion: air emboli, thrombus, tumor Pregnancy: aortocaval compression

Hemothorax

contusion

Anaphylaxis

Hepatic failure

Sepsis

Resuscitation from shock refers to the restoration of normal physiology after injury. Resuscitation after a hemorrhagic shock requires the restoration of a normal circulating

Early resuscitation occurs while active bleeding is still ongoing. The goals in early resuscitation are to maintain the systolic blood pressure at 80 to 100 mmHg, maintain the hematocrit at 25-30%, maintain the coagulation panel and ionized calcium level within a normal range, keep the platelet count > 50,000, maintain the core temperature at > 35 °C, and prevent worsening acidosis. Late resuscitation is defined as after the bleeding is controlled and has separate goals that include maintaining the systolic blood pressure > 100 mmHg, maintaining a normal hematocrit, normalizing the coagulation status, electrolytes,

blood volume, normal vascular tone, and normal tissue perfusion. (Miller, 2009)

temperature, and urine output, and maximizing the cardiac output. (Miller, 2009)

 Adrenal insufficiency Arteriovenous fistula

 Preexisting cardiac disease Myocardial infarction

Systemic inflammatory disease

cardiogenic, and vasodilated shock should be ruled out. (Smith, 2008)

**Obstructive**  Tension pneumothorax

**Cardiogenic**  Blunt cardiac injury: myocardial

**Vasodilated**  Spinal cord injury

**7. Intra-operative complications**

Table 28. Types of shock (Smith, 2008)

**7.1 Hypotension**

Patients with orthopedic injury and poly-trauma including orthopedic injury may present with a significant blood loss. Massive transfusion protocols (MTP) have been designed and implemented in many large urban trauma centers to provide a rapidly bleeding patient with automatic regular shipments of blood products to facilitate fluid resuscitation during an emergency surgery. The goal of the design ratio of packed red blood cells, plasma, and platelets built into the protocol are based on the need for volume support, oxygen delivery, and coagulation support required at each stage of the resuscitation. (Smith, 2008)

When should the MTP be initiated? This is often a judgment call made by the Surgeon or Anesthesiologist. It can be guided by these criteria: 5-unit blood loss in one hour, or 10-unit blood loss anticipated for the entire case or within 12-24 hours of observation, or hypovolemic hypotension uncorrected by crystalloid and/or packed red blood cell resuscitation during an ongoing hemorrhage. (Smith, 2008)

During the MTP, a cooler containing blood products is brought one at a time to the operating room as each is finished or until the MTP is terminated. The MTP will vary from hospital to hospital. See below for a sample MTP. (Smith, 2008)

Table 29. Sample massive transfusion protocol (MTP) (Smith, 2008)

#### **7.3 Hypothermia**

Hypothermia is defined as a core temperature less than 36 °C. Hypothermia is usually caused by prolonged exposures to cold temperatures. Many trauma patients present to the operating room very cold secondary to prolonged exposure to the elements after the initial trauma. Mild hypothermia, about 1-2 °C below normal triples the incidence of morbid cardiac outcomes, triples the incidence of wound infections, directly impairs immune function, prolongs hospitalization by up to 20%, significantly increases surgical blood loss, and can lead to cold-induced platelet dysfunction. (Lovich-Sapola, 2010)

Trauma patients may enter the operating room already cold, but even if they are normothermic, the patients' core temperatures usually decrease by 0.5-1.5 °C in the first 30 minutes after the induction of anesthesia secondary to the internal redistribution of heat. Mechanisms for heat loss include radiation loss, convection loss, conduction loss, and evaporative loss. (Lovich-Sapola, 2010)

Anesthesia for Orthopedic Trauma 211

During the active re-warming of a patient, the following monitors are routine: continuous core temperature monitor and bladder catheterization to monitor the urine output and therefore the adequacy of the intravascular volume and organ perfusion.  On occasion, a pulmonary artery catheter may be useful to follow the cardiac output (Lovich-

Fat embolism syndrome (FES) occurs when microembolism of fat and marrow from the patients long-bones result in clinically significant symptoms. Intra-operative transesophageal echocardiography (TEE) has shown that most patients undergoing longbone fracture manipulation experience this microembolization, but most patients do not have any clinical impact. Clinically significant FES occurs in about 3-10% of patients having a long-bone repair with the higher incidence if the patient has multiple long-bone fractures. "The presentation of FES can be gradual, developing over 12 to 72 hours, or fulminate

leading to acute respiratory distress and cardiac arrest." (Miller, 2009)

Mental status change: drowsiness, confusion, obtundation, coma

Table 32. Clinical manifestation for FES (Miller, 2009; Smith, 2008)

Petechial rash of the upper body: conjunctiva, oral mucosa, skin folds of the neck,

Lab results: fat microglobulinemia, anemia, thrombocytopenia, high ESR, fat globules in

**Treatment for Fat Embolism Syndrome (FES)**

Ventilation management: oxygen, higher PEEP, and possible long-term mechanical

Acute compartment syndrome is defined as a "condition in which increased pressure within a limited space compromises the circulation and function of the tissues within that space." The most common fractures associated with compartment syndrome are the tibial shaft and forearm. The most common cause of compartment syndrome with orthopedic trauma patients is edema secondary to muscle injury and associated hematoma formation.

**Clinical Manifestations of Fat Embolism Syndrome (FES)**

shoulders, axilla, upper arms, and chest Elevated pulmonary artery pressure

Chest X-ray: bilateral alveolar infiltrates

Table 33. Treatment for FES (Miller, 2009)

**7.5 Acute compartment syndrome**

Decreased cardiac index

Early recognition

ventilatory support Judicious fluid management

Sapola, 2010)

Hypoxia Tachycardia

the urine

(Miller, 2009)

**7.4 Fat embolism syndrome**

Table 30. Physiologic effects of hypothermia. (Lovich-Sapola, 2010)

#### **Treatment of Hypothermia**

Table 31. Treatment of hypothermia (Lovich-Sapola, 2010 and Smith, 2008)

33 °C Hypertension, tachycardia, and increased cardiac output Increased oxygen consumption

Significant neural and organ depression

28 °C Hyperglycemia: plasma fall in insulin and increased

Decreased oxygen consumption

Prolonged drug metabolism

 Ventricular fibrillation Cessation of respiration

Table 30. Physiologic effects of hypothermia. (Lovich-Sapola, 2010)

Body loses its ability to generate heat by shivering.

catecholamines from the stress of hypothermia

Oxy-hemoglobin dissociation curve shift to the left

Prolonged bleeding and clotting times; usually reversible with re-

Hypovolemia: cold diuresis; physiologic increase in hematocrit

 Cardiac arrhythmias: bradycardia, nodal rhythm, premature ventricular contractions, atrio-ventricular block, ventricular

EKG changes: prolonged PR interval, widening of the QRS

Acid-base disturbances, unless ventilation is maintained

**Treatment of Hypothermia**  Surface re-warming: warmed blankets, forced air heating blankets, gel pad warming

Extracorporeal: hemodialysis, hemofiltration, continuous arteriovenous re-warming,

Warm body cavity lavage: bladder, stomach, chest, and abdominal cavity

Table 31. Treatment of hypothermia (Lovich-Sapola, 2010 and Smith, 2008)

complex, prolonged QT interval, ST segment elevation, J wave

**Temperature Physiologic Effect**

Muscle mis-coordination

Loss of consciousness

and hypotension

fibrillation

 Thrombocytopenia Metabolic acidosis

<25°C Metabolism is reduced by 60%

Heated (humidified) inspired gases

Warming the operating room

cardiopulmonary bypass

system

Fluid warmers

Hyperventilation

 Shivering Piloerection

 Confusion 30 °C Metabolism is reduced by 50%

warming

During the active re-warming of a patient, the following monitors are routine: continuous core temperature monitor and bladder catheterization to monitor the urine output and therefore the adequacy of the intravascular volume and organ perfusion.  On occasion, a pulmonary artery catheter may be useful to follow the cardiac output (Lovich-Sapola, 2010)

#### **7.4 Fat embolism syndrome**

Fat embolism syndrome (FES) occurs when microembolism of fat and marrow from the patients long-bones result in clinically significant symptoms. Intra-operative transesophageal echocardiography (TEE) has shown that most patients undergoing longbone fracture manipulation experience this microembolization, but most patients do not have any clinical impact. Clinically significant FES occurs in about 3-10% of patients having a long-bone repair with the higher incidence if the patient has multiple long-bone fractures. "The presentation of FES can be gradual, developing over 12 to 72 hours, or fulminate leading to acute respiratory distress and cardiac arrest." (Miller, 2009)

Table 32. Clinical manifestation for FES (Miller, 2009; Smith, 2008)

Table 33. Treatment for FES (Miller, 2009)

#### **7.5 Acute compartment syndrome**

Acute compartment syndrome is defined as a "condition in which increased pressure within a limited space compromises the circulation and function of the tissues within that space." The most common fractures associated with compartment syndrome are the tibial shaft and forearm. The most common cause of compartment syndrome with orthopedic trauma patients is edema secondary to muscle injury and associated hematoma formation. (Miller, 2009)

Anesthesia for Orthopedic Trauma 213

Crush syndrome is defined as rhabdomyolisis secondary to the associated hypovolemia and toxin exposure after a crush injury with resultant skeletal muscle compression. Rhabdomyolysis occurs when the components of the damaged skeletal muscle enter the patient's circulation. Serum creatinine kinase (CK) levels correlate with the degree of muscle injury, and can be used to assess the severity of the rhabdomyolysis. Acute renal failure (ARF) is the most serious consequence of rhabdomyolysis. ARF "occurs in 4-33% of the

**Common Mechanisms of a Crush Injury**

Improper intra-operative positioning: extended lithotomy, lateral decubitus

**Clinical Symptoms of a Crush Injury**

**Treatment and Prevention of Renal Failure after a Crush Injury**

Confirm the urine flow prior to forced mannitol-alkaline diuresis (controversial

Table 37. Treatment and prevention of renal failure after a crush injury (Malinoski, 2004)

Vascular compromise: arterial thrombosis, embolus, traumatic injury

cases"and has a "mortality rate of 3-50%". (Malinoski, 2004)

 Alcohol intoxication: fall, immobility, coma Earthquakes, landslides, building collapses

Dark urine secondary to myoglobinuria

Early and vigorous volume replacement: crystalloid

Alkalization of the urine with sodium bicarbonate

The total body deficit may be up to 15 L

Sudden automobile deceleration

Prolonged tourniquet use

Soft tissue infections

 Swollen extremities Rhabdomyolysis

 Acute renal failure Electrolyte abnormalities

 Treat the hypovolemic shock Treat the hyperkalemia

Closely monitor the urine output Closely monitor the electrolytes

Shock

treatment) Mannitol

Electrical injuries: lightning strikes, high-voltage power lines

Trapped in one position for an extended period of time

 Medications: steroids, neuromuscular blocking drugs Table 35. Crush injury mechanisms (Malinoski, 2004; Miller, 2009)

Table 36. Clinical symptoms of a crush injury (Malinoski, 2004; Miller, 2009)

Goal: prevent the precipitation of myoglobin in the renal tubules

Daily hemodialysis or continuous hemodialysis/hemofiltration

**7.6 Crush injury**

Blunt trauma

The diagnosis of acute compartment syndrome is primarily clinical. (Olsen, 2005) The earliest symptoms are often "pain out of proportion to the injury", pain with passive motion, and tense swelling of the affected area. (Miller, 2009; Smith, 2008) Decreased distal sensation and loss of proprioception are seen next, followed by complete anesthesia and muscle weakness. (Smith, 2008) The late-onset "classic"symptoms of pulselessness, pallor, paralysis, and paresthesia often do not present until irreversible loss of function has occurred. (Miller, 2009; Olsen, 2005; Smith, 2008))

The Surgeon and the Anesthesiologist must be careful because certain anesthetic techniques such as nerve blocks and epidural anesthesia can mask these symptoms and lead to a delay in diagnosis. This delay can be limb-threatening. (Olsen, 2005) These same symptoms can also be masked by an altered level of consciousness, deep sedation, or large doses of pain medication; therefore a high index for suspicion is required along with serial physical examinations so that the compartment syndrome is not missed. (Miller, 2009; Smith, 2008)

The treatment for compartment syndrome is a fasciotomy of all involved compartments. To be effective, the fasciotomy must be performed as early as possible to prevent irreversible damage. (Miller, 2009)

Table 34. Conditions that can result in acute compartment syndrome (Miller, 2009; Olsen, 2005)

#### **7.6 Crush injury**

212 Orthopedic Surgery

The diagnosis of acute compartment syndrome is primarily clinical. (Olsen, 2005) The earliest symptoms are often "pain out of proportion to the injury", pain with passive motion, and tense swelling of the affected area. (Miller, 2009; Smith, 2008) Decreased distal sensation and loss of proprioception are seen next, followed by complete anesthesia and muscle weakness. (Smith, 2008) The late-onset "classic"symptoms of pulselessness, pallor, paralysis, and paresthesia often do not present until irreversible loss of function has

The Surgeon and the Anesthesiologist must be careful because certain anesthetic techniques such as nerve blocks and epidural anesthesia can mask these symptoms and lead to a delay in diagnosis. This delay can be limb-threatening. (Olsen, 2005) These same symptoms can also be masked by an altered level of consciousness, deep sedation, or large doses of pain medication; therefore a high index for suspicion is required along with serial physical examinations so that the compartment syndrome is not missed. (Miller, 2009; Smith, 2008)

The treatment for compartment syndrome is a fasciotomy of all involved compartments. To be effective, the fasciotomy must be performed as early as possible to prevent irreversible

**Conditions That Can Result in Acute Compartment Syndrome**

Table 34. Conditions that can result in acute compartment syndrome (Miller, 2009; Olsen,

occurred. (Miller, 2009; Olsen, 2005; Smith, 2008))

Fractures: tibial shaft, radius, ulna

 Infiltration form venous or arterial puncture sites Extravasation of fluids with pulsatile lavage

Intraosseous fluid replacement in a child/infant

Hemorrhage with hematoma formation

Drug overdoes: prolonged immobile state

damage. (Miller, 2009)

 Gunshot wounds Crush injury Burns Snake bites

 Pressurized infusions Casts and circular dressings

 Post-ischemic swelling Reperfusion injury

Prolonged limb compression

Bleeding disorders

Contusions

Trauma:

Iatrogenic:

Vascular:

2005)

Crush syndrome is defined as rhabdomyolisis secondary to the associated hypovolemia and toxin exposure after a crush injury with resultant skeletal muscle compression. Rhabdomyolysis occurs when the components of the damaged skeletal muscle enter the patient's circulation. Serum creatinine kinase (CK) levels correlate with the degree of muscle injury, and can be used to assess the severity of the rhabdomyolysis. Acute renal failure (ARF) is the most serious consequence of rhabdomyolysis. ARF "occurs in 4-33% of the cases"and has a "mortality rate of 3-50%". (Malinoski, 2004)

#### **Common Mechanisms of a Crush Injury**

Table 35. Crush injury mechanisms (Malinoski, 2004; Miller, 2009)

#### **Clinical Symptoms of a Crush Injury**

Table 36. Clinical symptoms of a crush injury (Malinoski, 2004; Miller, 2009)

#### **Treatment and Prevention of Renal Failure after a Crush Injury**

Early and vigorous volume replacement: crystalloid

Confirm the urine flow prior to forced mannitol-alkaline diuresis (controversial treatment)

Closely monitor the urine output

Closely monitor the electrolytes

Daily hemodialysis or continuous hemodialysis/hemofiltration

Table 37. Treatment and prevention of renal failure after a crush injury (Malinoski, 2004)

Anesthesia for Orthopedic Trauma 215

Opioids are the cornerstone of acute pain management. The most commonly used types of opioids include: morphine, meperidine, codeine, fentanyl, hydromorphone, oxycodone, methadone, and buprenorphine. Opioids significantly decrease pain score ratings in trauma patients. Carefully titrated opioids are used as the most common treatment method in trauma cases. The opioids are usually given by a fixed dosing schedule or PCA technique. Opioids can be administered by several routes (oral, intravenous, subcutaneous, intramuscular, intrathecal, epidural, transmucosal, transdermal, inhaled, intra-articular, and local injection into the wound) thus making them very versatile in clinical applications. Side effects of opioids include nausea, vomiting, constipation, sedation, altered sensorium, and

Ketamine, a phencyclidine derivative, is a noncompetitive inhibitor at the NMDA receptor. Ketamine is unique in that it produces a dissociative state that causes amnesia and an intense analgesia, while maintaining the vital brain stem functions and hemodynamic stability. It provides profound amnesia and analgesia. Ketamine has been shown to decrease opioid requirements. Ketamine is a very useful drug for frequent dressing changes, treating burn patients, and minor trauma procedures. The drawbacks of ketamine include resulting excessive secretions, agitation on recovery, hallucinations, and increase in intracranial and intraocular pressure. The hallucinations can be decreased by giving a benzodiazepine. Ketamine should be avoided in any patient with an associated head trauma. (Smith, 2008) Tricyclic antidepressants (TCAs) are useful in the treatment of neuropathic pain conditions. TCAs are especially helpful in trauma patients because they can reduce pain, facilitate sleep, and alleviate depression. TCAs have a delayed onset of action; the patient must be educated that it may take days to see results. Recommended TCAs include amitriptyline, nortriptyline, imiprimine, and desipramine. Side effects can include: prolonged QT interval,

Gabapentin and pregabalin are anticonvulsants used to treat neuropathic, postsurgical, and posttraumatic pain. These medications can only be given in the oral form, so they can not be started until the patient is tolerating food. Side effects may include sedation, dizziness,

Multimodal analgesia is the key to the treatment of pain in a poly-trauma patient. The goal is an optimal balance of multiple medications. In addition to pharmacologic methods, nonpharmacologic approaches such as psychologic support, counseling, hypnosis, relaxation

Regional analgesia from an epidural or continuous catheter should be considered in any orthopedic trauma patient if possible secondary to the resultant sparing of narcotics, high level of patient satisfaction, improved pulmonary function (with associated thoracoabdominal injury), and facilitated early mobilization. (Miller, 2009) Localized and regional pain can be treated with local, regional, or neuraxial analgesia. Regional blocks provide selective pain relief with fewer hemodynamic side effects than neuraxial blocks. Regional anesthesia can decrease the need for opioids and therefore result in decreased nausea, vomiting, and sedation. Regional anesthesia can also be used for surgical anesthesia as well

techniques, biofeedback, and acupuncture should be considered. (Smith, 2008)

respiratory depression. (Smith, 2008)

arrhythmia, and paralytic ileus. (Smith, 2008)

as post-operative pain management. (Smith, 2008)

confusion, and headache. (Smith, 2008)

**8.2 Regional anesthesia**

#### **8. Post-operative pain management**

Managing a patient's pain is always challenging, but it can be especially difficult in a polytrauma/multiple orthopedic injury patient. Pain management in these patients will require a multimodal approach. Inadequate analgesia can lead to complications with healing, immune function, and autonomic dysfunction. Prolonged untreated pain can lead to the development of a chronic pain state that can be very difficult to treat and may lead to lifelong problems. The goal of pain management in a trauma patient is to reduce the stress response, and provide the patient with pain relief while maintaining cardiovascular stability and tissue homeostasis. (Smith, 2008)

The initial pain management after an orthopedic surgery case usually begins in the recovery room. The administration of rapidly acting intravenous agents in small doses at frequent intervals under continuous monitoring should be continued until adequate pain relief is attained. The total dose of medication required to relieve the patient's pain can then be used to determine the patient's basal requirements before starting a long-acting medication or patient-controlled analgesia (PCA). (Miller, 2009)

Post-operative physical therapy after an orthopedic injury can have a significant impact on the pain medication requirements. It is important to consider the physical therapy schedule and plan the pain medication dosing appropriately. The goal of the dosing is to allow for enough pain relief to successfully perform the physical therapy but not so much that they are too sedated to participate. Physical therapy and activity after an orthopedic trauma is very important because it lowers the risk of pulmonary embolism, venous thrombosus, and decubitus ulcers. While physical therapy is more painful in the short term, the sooner the patient is mobilized, the lower the analgesic doses will be in the long term. (Miller, 2009)

Neuropathic pain occurs when there is direct injury to a major sensory nerve. This type of injury is common after spinal cord trauma, traumatic amputations, and major crush injuries. Neuropathic pain presents with a burning sensation, intermittent electrical shocks, and dysthesia in the affected dermatomal distribution. Neuropathic pain often responds poorly to narcotic analgesics used for somatic pain. The first line of treatment should be with gabapentin, 200 mg three times per day, with daily titration up to a maximum of 2-3 g/day. Persistent neuropathic pain may require selective regional anesthesia to "break the cycle" of spinal cord receptor recruitment. (Miller, 2009)

#### **8.1 Pharmacologic measures**

Acetaminophen is an analgesic/ antipyretic drug. It is popular because it is relatively safe compared with other anti-inflammatory medications. Acetaminophen is an important adjuvant to opioid analgesia because it decreases the total amount of opioid required and therefore decreases the adverse effects of excess opioids. The ceiling effect of oral acetaminophen is 1g/dose. (Smith, 2008)

Nonsteroidal anti-inflammatory drugs (NSAIDs) are powerful COX inhibitors and the first line treatment for many pain conditions. NSAIDs are effective for moderate to severe pain. The use of NSAIDs is often limited by their side effect profile: gastric ulcers, renal and platelet dysfunction, and decreased bone fusion and healing which is particularly troublesome in orthopedic trauma patients. (Smith, 2008)

Managing a patient's pain is always challenging, but it can be especially difficult in a polytrauma/multiple orthopedic injury patient. Pain management in these patients will require a multimodal approach. Inadequate analgesia can lead to complications with healing, immune function, and autonomic dysfunction. Prolonged untreated pain can lead to the development of a chronic pain state that can be very difficult to treat and may lead to lifelong problems. The goal of pain management in a trauma patient is to reduce the stress response, and provide the patient with pain relief while maintaining cardiovascular stability

The initial pain management after an orthopedic surgery case usually begins in the recovery room. The administration of rapidly acting intravenous agents in small doses at frequent intervals under continuous monitoring should be continued until adequate pain relief is attained. The total dose of medication required to relieve the patient's pain can then be used to determine the patient's basal requirements before starting a long-acting medication or

Post-operative physical therapy after an orthopedic injury can have a significant impact on the pain medication requirements. It is important to consider the physical therapy schedule and plan the pain medication dosing appropriately. The goal of the dosing is to allow for enough pain relief to successfully perform the physical therapy but not so much that they are too sedated to participate. Physical therapy and activity after an orthopedic trauma is very important because it lowers the risk of pulmonary embolism, venous thrombosus, and decubitus ulcers. While physical therapy is more painful in the short term, the sooner the patient is mobilized, the lower the analgesic doses will be in the long term. (Miller, 2009)

Neuropathic pain occurs when there is direct injury to a major sensory nerve. This type of injury is common after spinal cord trauma, traumatic amputations, and major crush injuries. Neuropathic pain presents with a burning sensation, intermittent electrical shocks, and dysthesia in the affected dermatomal distribution. Neuropathic pain often responds poorly to narcotic analgesics used for somatic pain. The first line of treatment should be with gabapentin, 200 mg three times per day, with daily titration up to a maximum of 2-3 g/day. Persistent neuropathic pain may require selective regional anesthesia to "break the cycle" of

Acetaminophen is an analgesic/ antipyretic drug. It is popular because it is relatively safe compared with other anti-inflammatory medications. Acetaminophen is an important adjuvant to opioid analgesia because it decreases the total amount of opioid required and therefore decreases the adverse effects of excess opioids. The ceiling effect of oral

Nonsteroidal anti-inflammatory drugs (NSAIDs) are powerful COX inhibitors and the first line treatment for many pain conditions. NSAIDs are effective for moderate to severe pain. The use of NSAIDs is often limited by their side effect profile: gastric ulcers, renal and platelet dysfunction, and decreased bone fusion and healing which is particularly

**8. Post-operative pain management**

and tissue homeostasis. (Smith, 2008)

patient-controlled analgesia (PCA). (Miller, 2009)

spinal cord receptor recruitment. (Miller, 2009)

acetaminophen is 1g/dose. (Smith, 2008)

troublesome in orthopedic trauma patients. (Smith, 2008)

**8.1 Pharmacologic measures**

Opioids are the cornerstone of acute pain management. The most commonly used types of opioids include: morphine, meperidine, codeine, fentanyl, hydromorphone, oxycodone, methadone, and buprenorphine. Opioids significantly decrease pain score ratings in trauma patients. Carefully titrated opioids are used as the most common treatment method in trauma cases. The opioids are usually given by a fixed dosing schedule or PCA technique. Opioids can be administered by several routes (oral, intravenous, subcutaneous, intramuscular, intrathecal, epidural, transmucosal, transdermal, inhaled, intra-articular, and local injection into the wound) thus making them very versatile in clinical applications. Side effects of opioids include nausea, vomiting, constipation, sedation, altered sensorium, and respiratory depression. (Smith, 2008)

Ketamine, a phencyclidine derivative, is a noncompetitive inhibitor at the NMDA receptor. Ketamine is unique in that it produces a dissociative state that causes amnesia and an intense analgesia, while maintaining the vital brain stem functions and hemodynamic stability. It provides profound amnesia and analgesia. Ketamine has been shown to decrease opioid requirements. Ketamine is a very useful drug for frequent dressing changes, treating burn patients, and minor trauma procedures. The drawbacks of ketamine include resulting excessive secretions, agitation on recovery, hallucinations, and increase in intracranial and intraocular pressure. The hallucinations can be decreased by giving a benzodiazepine. Ketamine should be avoided in any patient with an associated head trauma. (Smith, 2008)

Tricyclic antidepressants (TCAs) are useful in the treatment of neuropathic pain conditions. TCAs are especially helpful in trauma patients because they can reduce pain, facilitate sleep, and alleviate depression. TCAs have a delayed onset of action; the patient must be educated that it may take days to see results. Recommended TCAs include amitriptyline, nortriptyline, imiprimine, and desipramine. Side effects can include: prolonged QT interval, arrhythmia, and paralytic ileus. (Smith, 2008)

Gabapentin and pregabalin are anticonvulsants used to treat neuropathic, postsurgical, and posttraumatic pain. These medications can only be given in the oral form, so they can not be started until the patient is tolerating food. Side effects may include sedation, dizziness, confusion, and headache. (Smith, 2008)

Multimodal analgesia is the key to the treatment of pain in a poly-trauma patient. The goal is an optimal balance of multiple medications. In addition to pharmacologic methods, nonpharmacologic approaches such as psychologic support, counseling, hypnosis, relaxation techniques, biofeedback, and acupuncture should be considered. (Smith, 2008)

#### **8.2 Regional anesthesia**

Regional analgesia from an epidural or continuous catheter should be considered in any orthopedic trauma patient if possible secondary to the resultant sparing of narcotics, high level of patient satisfaction, improved pulmonary function (with associated thoracoabdominal injury), and facilitated early mobilization. (Miller, 2009) Localized and regional pain can be treated with local, regional, or neuraxial analgesia. Regional blocks provide selective pain relief with fewer hemodynamic side effects than neuraxial blocks. Regional anesthesia can decrease the need for opioids and therefore result in decreased nausea, vomiting, and sedation. Regional anesthesia can also be used for surgical anesthesia as well as post-operative pain management. (Smith, 2008)

Anesthesia for Orthopedic Trauma 217

**Lumbar Plexus**  Patients with unilateral lower limb trauma that are

Tibial plateau fracture

Tibial plateau fracture

**Saphenous Nerve**  Medial aspect of the foot (ankle and forefoot

**Sciatic Nerve**  Analgesia to the posterior compartment of the thigh

IV regional anesthesia is a simple, reliable, cost effective block that is good for short operative procedure on an extremity. Lidocaine is the recommended drug for this block, secondary to it being less cardiotoxic than bupivacaine. IV regional anesthesia requires exsanguination of the limb with a tight bandage, which may be very uncomfortable to an injured limb. Early deflation of the cuff used to keep the local anesthesia in the affected

Epidural anesthesia has been shown to provide superior dynamic analgesia for the treatment of post-operative hip fracture pain during rehabilitation. The patients are significantly less restricted by their pain. However, studies have shown that this decrease in pain does not significantly translate into enhanced rehabilitation. (Foss, 2005) Again, patients undergoing a hip fracture surgery have the highest risk of death from a pulmonary embolism. A spinal anesthetic is recommended for the actual surgery, but an epidural is rarely indicated for post-operative pain management secondary to the aggressive post-

Analgesia to most of the lower leg

Must be combined with the sciatic nerve block

 Femur fracture Hip fracture

ACL repair

**Fascia Iliaca**  Same indications as a femoral nerve block

**Ankle**  Surgical procedures of the foot

Table 39. Lower limb blocks (Miller, 2009; Smith, 2008)

**8.2.3 Intravenous (IV) regional anesthesia (Bier's block)**

extremity can result in local anesthetic toxicity. (Smith, 2008)

operative anticoagulation that will be initiated. (Miller, 2009)

**8.2.4 Epidural anesthesia**

fractures) Tourniquet pain

not candidates for an epidural

**Lower Limb Blocks Indication**

**Femoral Nerve**  Femoral shaft fracture

**8.2.2 Lower limb blocks**

Prior to any block, the Anesthesiologist must first evaluate the patient. A review of the patient's medical history including an evaluation for preexisting neurologic injuries, medication history, capability to give informed consent, ability to cooperate for the procedure, availability of landmarks, ability to properly position the patient for the procedure, laboratory values (coagulation studies and platelet count), and finally the approval of the Surgeon to perform the block. A block has the potential to delay the diagnosis of a compartment syndrome and potentially worsen existing nerve damage, and therefore may not be safe in certain patients. (Smith, 2008)

Ultrasound guidance is recommended for the placement of any regional block, but especially in trauma patients, secondary to the maximal benefit of patient safety and comfort. The peripheral nerve stimulation technique can be painful and should be avoided. (Smith, 2008)

#### **8.2.1 Upper limb blocks**

Table 38. Upper limb blocks (Miller, 2009; Smith, 2008)

#### **8.2.2 Lower limb blocks**

216 Orthopedic Surgery

Prior to any block, the Anesthesiologist must first evaluate the patient. A review of the patient's medical history including an evaluation for preexisting neurologic injuries, medication history, capability to give informed consent, ability to cooperate for the procedure, availability of landmarks, ability to properly position the patient for the procedure, laboratory values (coagulation studies and platelet count), and finally the approval of the Surgeon to perform the block. A block has the potential to delay the diagnosis of a compartment syndrome and potentially worsen existing nerve damage, and

Ultrasound guidance is recommended for the placement of any regional block, but especially in trauma patients, secondary to the maximal benefit of patient safety and comfort. The peripheral nerve stimulation technique can be painful and should be avoided.

**Upper Limb Blocks Indication Concerns**

**Supraclavicular block**: injuries below the shoulder **Infraclavicular block**: injury below the shoulder; better for continuous catheter placement and in patients with cervical injury because the head may stay in a neutral position during

Attractive for patients with coagulopathy secondary to the compressibility of the vessels in this location

nerves can be blocked at the

upper arm

placement

**Axillary Brachial Plexus** Hand and forearm trauma

**Wrist** Radial, median, and ulnar

Table 38. Upper limb blocks (Miller, 2009; Smith, 2008)

wrist

Injuries of the shoulder and

 Associated with 100% incidence of diaphragm paresis which can last

> patients with a cervical injury because of difficulty in positioning

up to 6 hours Difficult to place in

 Potential risk for pneumothorax

Must be able to abduct

 Failure to anesthetize the musculocutaneous

the arm

nerve

therefore may not be safe in certain patients. (Smith, 2008)

(Smith, 2008)

**Plexus**

**8.2.1 Upper limb blocks**

**Interscalene Brachial**

**Brachial Plexus Around**

**the Clavicle**

Table 39. Lower limb blocks (Miller, 2009; Smith, 2008)

#### **8.2.3 Intravenous (IV) regional anesthesia (Bier's block)**

IV regional anesthesia is a simple, reliable, cost effective block that is good for short operative procedure on an extremity. Lidocaine is the recommended drug for this block, secondary to it being less cardiotoxic than bupivacaine. IV regional anesthesia requires exsanguination of the limb with a tight bandage, which may be very uncomfortable to an injured limb. Early deflation of the cuff used to keep the local anesthesia in the affected extremity can result in local anesthetic toxicity. (Smith, 2008)

#### **8.2.4 Epidural anesthesia**

Epidural anesthesia has been shown to provide superior dynamic analgesia for the treatment of post-operative hip fracture pain during rehabilitation. The patients are significantly less restricted by their pain. However, studies have shown that this decrease in pain does not significantly translate into enhanced rehabilitation. (Foss, 2005) Again, patients undergoing a hip fracture surgery have the highest risk of death from a pulmonary embolism. A spinal anesthetic is recommended for the actual surgery, but an epidural is rarely indicated for post-operative pain management secondary to the aggressive postoperative anticoagulation that will be initiated. (Miller, 2009)

Anesthesia for Orthopedic Trauma 219

Every patient requires a preoperative evaluation that is complete as possible in the time allowed. Airway management may be difficult secondary to the potential for associated cervical spine injuries, and the proper preparation and precautions should be taken. Every patient requires the standard ASA monitors for the procedure, whether the orthopedic procedure is performed in the operating room, emergency room, or angiography suite.

The anesthetic technique should be chosen after careful evaluation of the risks and benefits of regional versus general anesthesia. It is important to remember that often a combination

The Anesthesiologist and the Orthopedic surgeon should be prepared for intra-operative complications including hypotension, blood loss, hypothermia, fat embolism, and compartment syndrome. Vigilance for these potential complications will lead to early

Finally, post-operative pain management also requires a multi-modal approach for the best results. Consider consulting the hospital's Acute Pain Management team for assistance. Untreated pain can lead to chronic pain syndromes and delayed rehabilitation in the

American Society of Anesthesiologists Committee on Standards and Practice Parameters.

Bassett M.D., Smith C.E. (2011). General Anesthesia for Trauma. In: Essentials of Trauma

Chung F. (2008). STOP Questionnaire. A Tool to Screen Patients for Obstructive Sleep

Fayad, A., Woo M. (2011). Echocardiography in Trauma. In: Essentials of Trauma

Fleisher, L.A., Beckman, J.A., Brown, K.A., et al. (2007). ACC/AHA 2007 Guidelines on the

Foss, N.B., Kristensen, M.T., Kristensen, B.B., et al. (2005). Effect of Postoperative Epidural

Lovich-Sapola, J.A. (2010). *Anesthesia Oral Board Review: Knocking Out the Boards.* Cambridge

Malinoski, D.J., Slater, M.S., Mullins, R.J. (2004). Crush Injury and Rhabdomyolysis. *Critical*

Miller, R.D., Eriksson, L.I., Fleisher, L.A., et al. (2009). *Miller: Miller's Anesthesia (7th ed.)*,

Executive Summary. *Circulation.*Vol. 116, (October 2007).

University Press, ISBN 978-0-521-75619, Cambridge

*Care Clinics.* Vol. 20, (2004), pp. 171-92.

Churchill Livingstone, New York.

Perioperative Cardiovascular Evaluation and Care for Noncardiac Surgery:

Analgesia on Rehabilitation and Pain after Hip Fracture Surgery: A Randomized, Double-blind, Placebo controlled Trial. *Anesthesiology.* Vol. 102, No. 6, (June 2005),

(2011). Practice Guidelines for Preoperative Fasting and the Use of Pharmacologic Agents to Reduce the Risk of Pulmonary Aspiration: Application to Healthy Patients Undergoing Elective Procedures. *Anesthesiology,* Vol. 114, No. 3, (March

diagnosis and treatment, and therefore a potentially improved outcome.

of both will yield the best results.

orthopedic injury patient.

2011), pp. 495-511.

pp. 1197-204.

Anesthesia, Cambridge. In Press

Anesthesia, Cambridge. In Press

Apnea. Anesthesiology 2008; 108:812–21

**10. References**

#### **8.2.5 Complications of regional anesthesia**

Table 40. Complications of regional anesthesia (Smith, 2008)

#### **9. Conclusion**

Orthopedic trauma requires a coordinated approach between the Surgeon and the Anesthesiologist. Orthopedic cases can often be some of the most complex for the Anesthesiologist secondary to the associated co-morbidities of the patient and the potential for multiple simultaneous injuries.

**Vascular Injury**  A risk/ benefit analysis is necessary in all patients:

and sensory/ motor deficit

the placement of any block **Diaphragm Weakness**  The phrenic nerve lies close to the interscalene groove

brachial plexus)

or a pneumothorax **Pneumothorax**  Serious complication of the supraclavicular brachial

structure

**Infections**  No reported cases in single injections

Table 40. Complications of regional anesthesia (Smith, 2008)

**9. Conclusion**

for multiple simultaneous injuries.

hours after the procedure. **Intravascular Injection**  Most nerve plexuses are in the vicinity of a vascular

**Horner's Syndrome**  The cervical sympathetic chain is in close proximity to

conjunctival engorgement

supraclavicular approaches

Increased risk with catheters

Orthopedic trauma requires a coordinated approach between the Surgeon and the Anesthesiologist. Orthopedic cases can often be some of the most complex for the Anesthesiologist secondary to the associated co-morbidities of the patient and the potential

platelet count, are on blood thinners

special concern if they are coagulopathic, have a low

 Evolution of neurologic deficits from an expanding hematoma can take 4 hours to 3 days to manifest itself Lack of treatment of a hematoma can lead to axon loss

Consider pre-existing trauma or vascular injury before

 Unilateral diaphragm weakness is not usually significant in healthy patients, but it can be devastating in patients with chest trauma, poor pulmonary status,

 Rare after the interscalene and infraclavicular approach Symptoms can be delayed and present as long as 6-12

Careful vigilance, multiple aspirations, and slow

The addition of epinephrine to the local anesthetic helps

the brachial plexus and can lead to Horner's syndrome Symptoms: ptosis, miosis, anhydrosis, and unilateral

Rate can be as high as 90% with interscalene and

plexus block and intercostal nerve block

Intravascular injections can lead to convulsions

to detect accidental intravascular injection

fractionated injections are important

(easily blocked by the interscalene approach to the

**8.2.5 Complications of regional anesthesia**

Every patient requires a preoperative evaluation that is complete as possible in the time allowed. Airway management may be difficult secondary to the potential for associated cervical spine injuries, and the proper preparation and precautions should be taken. Every patient requires the standard ASA monitors for the procedure, whether the orthopedic procedure is performed in the operating room, emergency room, or angiography suite.

The anesthetic technique should be chosen after careful evaluation of the risks and benefits of regional versus general anesthesia. It is important to remember that often a combination of both will yield the best results.

The Anesthesiologist and the Orthopedic surgeon should be prepared for intra-operative complications including hypotension, blood loss, hypothermia, fat embolism, and compartment syndrome. Vigilance for these potential complications will lead to early diagnosis and treatment, and therefore a potentially improved outcome.

Finally, post-operative pain management also requires a multi-modal approach for the best results. Consider consulting the hospital's Acute Pain Management team for assistance. Untreated pain can lead to chronic pain syndromes and delayed rehabilitation in the orthopedic injury patient.

#### **10. References**

Nahm, N.J., Como, J.J., Wilber, J.H., et al. (2011). Early Appropriate Care: Definitive

Patients With Multiple Injuries. *The Journal of Trauma.* [Epub ahead of print] Olsen, S.A. & Glascow, R.R. (2005) Acute Compartment Syndrome in Lower Extremity

Practice Guidelines for Perioperative Transesophageal Echocardiography. (2010). An

Robitaille, A., Williams, S., Tremblay, M.H., et al. (2008). Cervical Spine Motion During

Smith, C.E. (2008). *Trauma Anesthesia.* Cambridge University Press, ISBN 978-0-521-87058-0,

Vallier, H.A., Cureton, B.A., Ekstein, C., et al. (2010). Early Definitive Stabilization of

Echocardiography. Anesthesiology 2010; 112:1084 –96

*Trauma.* Vol. 69, No. 3, (September 2010), pp. 677-84.

7, (November 2005), pp. 436-4.

2008), pp. 935-41.

Cambridge.

Stabilization of Femoral Fractures Within 24 Hours of Injury Is Safe in Most

Musculoskeletal Trauma. *Journal of the Acadamy of Orthopeadic Surgeons.* Vol. 1, No.

Updated Report by the American Society of Anesthesiologists and the Society of Cardiovascular Anesthesiologists Task Force on Transesophageal

Tracheal Intubation with Manual In-Line Stabilization: Direct Laryngoscopy versus Glidesope Videolaryngoscopy. *Anesthesia and Analgesia.*Vol. 106. No. 3, (March

Unstable Pelvis and Acetabulum Fractures Reduces Morbidity. *The Journal of*

## *Edited by Zaid Al-Aubaidi and Andreas Fette*

Orthopaedic surgery is the widest and the strongest growing surgical specialty. It is clear, that the process of improving treatments and patients care, requires knowledge, and this requires access to studies, expert opinion and books. Unfortunately, the access to this knowledge is being materialized. As we believe that access to the medical knowledge should be reachable to everyone free of charge, this book was generated to cover the orthopaedic aspect. It will provide the reader with a mix of basic, but as well highly specialized knowledge. In the process of editing this book, my wife Jurgita has been, as usual, the most supportive person. I would like to thank her for being in my life. I would like to thank Mr. Greblo, the Publishing Process Manager, for all his help and last but not least thanks to our readers, as without them this book would have no meaning.

Orthopedic Surgery

Orthopedic Surgery

*Edited by Zaid Al-Aubaidi and Andreas Fette*

Photo by santypan / iStock


**Expression of Selenoprotein Genes and Association with Selenium Status in Colorectal Adenoma and Colorectal Cancer**

#### **David J. Hughes 1,\*, Tereza Kunická 2, Lutz Schomburg 3, Václav Liška 2,4, Niall Swan 5 and Pavel Souˇcek 2,4**

Received: 10 September 2018; Accepted: 14 November 2018; Published: 21 November 2018

**Abstract:** Dietary selenium (Se) intake is essential for synthesizing selenoproteins that are important in countering oxidative and inflammatory processes linked to colorectal carcinogenesis. However, there is limited knowledge on the selenoprotein expression in colorectal adenoma (CRA) and colorectal cancer (CRC) patients, or the interaction with Se status levels. We studied the expression of seventeen Se pathway genes (including fifteen of the twenty-five human selenoproteins) in RNA extracted from disease-normal colorectal tissue pairs, in the discovery phase of sixty-two CRA/CRC patients from Ireland and a validation cohort of a hundred and five CRC patients from the Czech Republic. Differences in transcript levels between the disease and paired control mucosa were assessed by the Mann-Whitney U-test. *GPX2* and *TXNRD3* showed a higher expression and *GPX3*, *SELENOP*, *SELENOS*, and *SEPHS2* exhibited a lower expression in the disease tissue from adenomas and both cancer groups (*p*-values from 0.023 to <0.001). In the Czech cohort, up-regulation of *GPX1*, *SELENOH*, and *SOD2* and down-regulation of *SELENBP1*, *SELENON*, and *SELENOK* (*p*-values 0.036 to <0.001) was also observed. We further examined the correlation of gene expression with serum Se status (assessed by Se and selenoprotein P, SELENOP) in the Irish patients. While there were no significant correlations with both Se status markers, *SELENOF*, *SELENOK*, and *TXNRD1* tumor tissue expression positively correlated with Se, while *TXNRD2* and *TXNRD3* negatively correlated with *SELENOP*. In an analysis restricted to the larger Czech CRC patient cohort, Cox regression showed no major association of transcript levels with patient survival, except for an association of higher *SELENOF* gene expression with both a lower disease-free and overall survival. Several selenoproteins were differentially expressed in the disease tissue compared to the normal tissue of both CRA and CRC patients. Altered selenoprotein expression may serve as a marker of functional Se status and colorectal adenoma to cancer progression.

**Keywords:** selenium (Se), selenoproteins; gene expression; selenium status; selenoprotein P; colorectal neoplasm; colorectal cancer; colorectal adenoma; biomarkers; cancer risk

#### **1. Introduction**

Worldwide, colorectal cancer (CRC) is the second most common cancer in women and the third in men, with highest prevalence in developed countries [1]. Modifiable dietary and lifestyle patterns are important contributors to CRC etiology [2].

In humans, the essential micronutrient selenium (Se) is incorporated as the amino acid selenocysteine in twenty-five selenoproteins, of which several are involved in a wide variety of metabolic pathways implicated in carcinogenesis [3–5]. Observational and intervention studies suggest that Se status can influence colorectal adenoma (CRA) and colorectal cancer (CRC) development risk, particularly in the geographical areas of suboptimal Se availability, such as much of Europe [6,7]. Transcriptomic and proteomic studies demonstrate that Se intake can influence the pattern of selenoprotein expression and biosynthesis, affecting numerous oxidative stress, inflammatory, and signal translation pathways that are important in colorectal carcinogenesis [8–10]. Due to the hierarchical pattern of organ-specific selenoprotein expression in conditions of limited Se supply [3,11], tumor tissue specific expression patterns may provide a biologically informative marker of CRC risk, especially in relation to inadequate Se status [8,12].

However, there is limited data on the selenoprotein expression in CRA and CRC patient tissue, or the relationship with Se status. Human studies have generally only examined gene or protein colonic carcinoma tissue expression of key antioxidant selenoprotein P (SELENOP), glutathione peroxidase (GPX), and thioredoxin reductase (TXNRD) selenoenzymes that have different hierarchical expression regulation depending on dietary Se [3,8]. These studies have generally shown overlapping patterns of expression, with some differences in areas of lower [13,14] to higher [15] Se availability.

To examine the pattern of selenoprotein gene expression in CRA and CRC disease tissue and the surrounding non-neoplastic mucosa, the mRNA transcript expression level of seventeen Se pathway genes (including fifteen selenoproteins, the related *SELENBP1* Se biosynthesis gene, and the interacting *SOD2* antioxidant gene [16]) was evaluated for one hundred and sixty-seven Irish and Czech individuals (forty CRA and a hundred and twenty-seven CRC patients) by quantitative real-time PCR (qPCR). We were further able to compare the gene expression levels with two serum Se status markers (Se and selenoprotein P; SELENOP) in the Irish cohort. Potentially, selenoprotein mRNA level may be an easily measured molecular biomarker for assessing the biologically relevant Se status [17] for protection of healthy colonic tissue.

#### **2. Materials and Methods**

#### *2.1. Patient Characteristics*

This project consisted of a discovery and validation phase. In the discovery set of samples (Irish cohort), paired tumor tissue and adjacent non-neoplastic mucosa samples (and matched sera, where available) were obtained from a total of sixty-two patients with colorectal carcinoma (*n* = 22) or adenoma (*n* = 40), diagnosed at the Departments of Gastroenterology and Surgery, The Adelaide and Meath Hospital, in Dublin. The validation set (Czech cohort) comprised a hundred and five colorectal carcinoma patients, diagnosed at the Department of Surgery, Teaching Hospital and Medical School, in Pilsen, between January 2008 and November 2011. The clinical characteristics of our study cohorts are summarized in Table 1.

Irish tissue samples were collected during resection of primary tumor or by biopsy, before treatment, while adenoma biopsies were obtained at colonoscopy during a pilot CRC screening program as described previously [18]. Only advanced CRAs were included in the study—forty tubular or tubulovillous adenomas of at least 1cm, of which fourteen had high grade dysplasia (HGD), as the most screen-relevant lesions [19]. Blood samples were collected immediately within one day of surgery or colonoscopy, in plain 6 mL VACUTAINER® tubes, (Cruinn Diagnostics, Dublin, Ireland) containing no anticoagulant. Blood was centrifuged at 2000× *g* for 10 min, within 4 h of collection, to separate the serum layer, which was then stored at −80 ◦C in cryovials. Collection and pathological processing of

tissue samples and retrieval of data acquisition of the Czech samples, was performed as previously described [20]. Histology was verified by an experienced pathologist at each center. All CRCs were classified according to the tenth revision of the International Classification of Diseases (ICD-10) and the second revision of the International Classification of Disease for Oncology (ICDO-2). The clinical data, including age at diagnosis, sex, pTNM (Tumor stage, Regional lymph node involvement, and distant metastasis) staging, histological grade of the tumor, and primary tumor localization were taken from patient medical records (see Table 1).

**Table 1.** Clinical-pathological characteristics of the studied cohort of patients.

SD = standard deviation; CRC = colorectal cancer; CRA = colorectal adenoma; - = not applicable or missing; TNM staging = Tumor stage, Regional lymph node involvement, and distant metastasis.

All patients were asked to carefully read and sign an informed consent, in accord with the 1964 Helsinki Declaration. The study was approved by the Ethical Committee of the St. James's Hospital and Federated Dublin Voluntary Hospitals Joint Research Ethics Committee (Ireland, reference 2007-37-17), and the Ethical Committee of the Medical Faculty and Teaching Hospital in Pilsen (Czech Republic, reference NT12025-4/2011). All samples were coded to protect patient anonymity.

#### *2.2. Isolation of Total RNA*

In the Irish cohort, the tissue samples were lysed on ice in 400 uL of lysis buffer (50 mmol/L HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.5; 150 mmol/L NaCl; 5 mmol/L EDTA) and protease inhibitor (Calbiochem, Hampshire, UK) followed by sonication (3 × 30 s, on ice) and centrifugation (10,000× *g* for 10 min at 4 ◦C). Total RNA (as well as gDNA and protein) was then extracted using the Norgen All-in-one purification kit (Norgen, Thorold, ON, Canada). In the Czech cohort, tissue samples were homogenized by a mechanical disruption in liquid nitrogen using mortar and pestle, and the total RNA was isolated using the Trizol Reagent according to the manufacturer protocol (Invitrogen, Carlsbad, CA, USA). RNA was then stored at minus 80 ◦C and the quantity and quality were measured, as previously described [21].

#### *2.3. Synthesis of Complementary DNA (cDNA)*

Reverse transcription of the total RNA (0.5 μg for each reaction) was performed, using random hexamer primers and the RevertAidTM First Strand cDNA Synthesis Kit (MBI Fermentas, Vilnius, Lithuania). Quality of the cDNA, in terms of DNA contamination, was confirmed by PCR amplification of *Ubiquitin C* [22].

#### *2.4. Relative Quantification of Gene Expression*

qPCR was performed using ViiA7 Real-Time PCR System, using TaqMan® Universal Master Mix and TaqMan® Gene Expression Assays (Life Technologies, Carlsbad, CA, USA) optimized primer and probe sets for the fifteen selenoprotein genes *SELENOP*, *SELENOS*, *GPX1-4*, *SELENOF*, *SEPHS2*, *SELENOH*, *SELENOK*, *SELENON*, *SELENOW*, *TXNRD1-3*, plus the Se biosynthesis gene *SELENBP1* and the interacting *SOD2* gene, in oxidative defense (Supplementary Table S1).

*POLR2A*, *PSMC4*, and *MRPL19* were used as the reference genes, based on stability assessment of twenty-four potential endogenous control genes in a test set of ten pairs of CRC tumors and non-neoplastic tissue samples [20]. Efficiency of the qPCR was determined for each assay using calibration curves (six-point, five times dilution), which were prepared from one non-neoplastic sample for the Czech cohort and from the mix of samples for the Irish cohort, respectively. The non-template control contained nuclease-free water instead of cDNA. Samples with variation larger than 0.5 Cq (quantitation cycle) were reanalyzed. The qPCR study adhered to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) Guidelines [23].

#### *2.5. Serum Selenium and Selenoprotein P Measurements*

The sample type (cancer, adenoma, and control) was blinded. Concentrations of the total Se were measured in 4 μL of each serum sample, using a bench-top total reflection X-ray fluorescence (TXRF) spectrometer (PicofoxTM S2, Bruker Nano GmbH, Berlin, Germany), as described previously [7]. SELENOP levels were ascertained by a colorimetric enzyme-linked immunoassay (SelenotestTM, ICI GmbH, Berlin, Germany) using 5 μL of each serum sample in a 1:21 dilution, according to the manufacturer's instructions [24]. Duplicate samples with differences in the Se concentration varying by more than 10% were measured again. The evaluation was performed with the GraphPad Prism 6.01 (GraphPad Software, La Jolla, CA, USA), using a four-parameter logistic function. The samples were measured in duplicate, and the mean concentration values, standard deviation (SD), and coefficients of variation (CVs) were calculated. The CVs were below 10% for the *SELENOP* controls 1 (1.5 mg/L) and 2 (8.6 mg/L) throughout the measurements.

#### *2.6. Statistical Analyses*

Transcript levels were analyzed by Viia7 System Software (Life Technologies, Carlsbad, CA, USA) and normalized levels of target genes (ratio of target gene Cq to the mean Cq of reference genes) were used for further statistical analyses, using SPSS v16.0 Software (SPSS Inc., Chicago, IL, USA). Differences in transcript levels between tumor and control tissues were assessed by nonparametric Mann-Whitney U-test. Nonparametric tests (the Kruskal-Wallis, the Mann-Whitney, and the Spearman's test) were also used for evaluation of associations of transcript levels with clinical data and other variables (Table 1). Kaplan-Meier Log Rank test and multivariate Cox regression analyses, adjusted to stage, surgical radicality, and chemotherapy, were used to assess the gene transcript levels, with subject disease-free survival (DFS) and overall survival (OS), in the Czech CRC cohort. Cox regression was used to determine the hazard ratio (HR) and the 95% confidence intervals (95% CI) of the transcript levels, were divided according to the median, with these survival outcomes.

Analyses were conducted using SPSS v16.0 Software (SPSS Inc., Chicago, IL, USA). All *p*-values were obtained from two-sided tests; *p*-values lower than 0.05 were considered statistically significant. Multiple testing adjustment (*PADJ*) was performed by the Benjamini-Hochberg correction (BH).

#### **3. Results**

#### *3.1. Selenoprotein Gene Transcript Levels in the Colorectal Adenomas*

In the Irish cohort, CRA patients showed a strongly significant lower expression of *SELENOP*, *SELENOS*, *GPX3*, and *SEPHS2* and higher expression levels of *GPX2*, *SELENOH*, and *TXNRD3* (*p* < 0.001 for all genes), in the disease tissue compared to the non-neoplastic control tissues. We tested the trend for expression with adenoma progression, compared to the normal tissues, by splitting the CRAs into two groups of twenty-six tubular and/or villous adenomas and another of fourteen HGDs. This suggested a declining expression of *SELENOP*, *SELENOS*, *GPX3*, and *SEPHS2* (respective *p*-values for trend at least 5.1 × 10−5) and increasing expression of *GPX2*, *SELENOH*, and *TXNRD3* (respective *p*-values at least 7 × 10−5), from the control to the increasingly dysplastic tissues.

#### *3.2. Selenoprotein Gene Transcript Levels in Colorectal Cancers*

In the tumor tissues of the CRC patients from Ireland, compared to the matched controls, *GPX2*, *GPX4*, *TXNRD3*, and *SOD2* were up-regulated (*p* = 0.023, 0.039, 0.003, and 0.036, respectively), while *SELENOP*, *SELENOS*, *GPX3*, *SEPHS2*, *SELENBP1*, and *SELENOK* were considerably down-regulated (*p* = 0.001, <0.001, <0.001, 0.002, <0.001, and 0.015, respectively).

In the validation study (Czech cohort), while many of the above observations were replicated, several disparities were observed. The Irish patients showed overexpressed levels of *GPX4* in tumors, compared to the control tissues, while the Czech subjects had no difference. Up-regulation of *GPX1* and *SELENOH* (*p* = 0.001 for both genes), and down-regulation of *SELENON* (*p* = 0.001) were demonstrated in the Czech patients, but no significant changes in the expression levels of these genes were found in the Irish cohort (although *SELENOH* was significantly more highly expressed in the adenoma tissue from the Irish CRA patients, *p* < 0.001).

All statistically significant correlations from the three groups of patients are summarized in Table 2. Directions of expression (higher or lower) were fully consistent for significant changes in the same gene across the different patient groups from Ireland and the Czech Republic. Gene expression levels did not correlate with the RNA integrity number (RIN), suggesting that RNA quality did not significantly influence the results.

Abbreviations: ↑ = higher expression in the neoplastic compared to the normal tissue, ↓ = lower expression in the neoplastic compared to the normal tissue, NS = not significant, TCGA = The Cancer Genome Atlas (https: //cancergenome.nih.gov/), COAD = colon adenocarcinoma, and READ = rectum adenocarcinoma. For the seventeen genes tested in the Irish and the Czech studies, only *SELENOF*, *SELENOW*, *TXNRD1*, and *TXNRD2* were not significantly different in at least in one of the neoplasm groups. Genes significant in all three Irish and Czech sample sets (*GPX2*, *GPX3*, *SELENOP*, *SELENOS*, *SEPHS2*, and *TXNRD3*) are marked by a dark background. *p*-values are in brackets. TCGA data: Analysis done on the TCGA tumor-normal data, using the GEPIA (Gene Expression Profiling Interactive Analysis) tool (http://gepia.cancer-pku.cn/about.html; Tang et al. Nucleic Acids Res. 2017; 45(W1): W98–W102).

There were no major consistent differences in the expression patterns by age, sex, or tumor sub-site (colon/rectum) across the Irish and the Czech CRC samples. Stratified analyses—in the larger Czech CRC group—of expression by tumor stage, grade, and TNM status, showed no significant differences after the BH correction, except for an association of a higher cancer stage and presence of lymph node metastasis with decreased *SOD2* expression (*PADJ* = 0.004 and *PADJ* = 0.048, respectively). The presence of lymph node metastasis also significantly associated with decreased *GPX1* expression

(*PADJ* = 0.048). As our data indicated that increasing numbers of selenoprotein genes were dysregulated from the adenoma to tumor tissue and, thus, possibly to be involved in disease progression, we examined gene expression with cancer patient outcome. We performed analyses of tumor selenoprotein transcript levels, divided by the median, with the DFS and the OS, using 8–76 months patient follow-up data that were available for ninety-eight of the Czech CRCs (mean values of 58 and 68 months, respectively). Only a higher *SELENOF* expression was associated with both a poorer DFS and OS, in a Kaplan-Meier (see Figure 1) and multivariate-adjusted Cox regression analysis (HR: 2.41; 95% CI: 1.08, 5.41; *p* = 0.032 for DFS, and HR: 4.13; 95% CI: 1.10, 15.39; *p* = 0.035 for OS). However, this result, although biologically-plausible based on previous data of the oncogenic potential of *SELENOF* [3], should be cautiously considered, as the CIs were wide and the point estimates lost significance after multiple testing correction. Additionally, *SELENOF* did not show significant differences in overall expression levels between the neoplastic and the matched mucosal tissues.

**Figure 1.** Association of the Selenoprotein F (*SELENOF*) gene expression with disease-free survival and overall survival, in colorectal cancer patients. Kaplan-Meier curves showing disease free survival (DFS) overall survival (OS) of 98 colorectal cancer (CRC) patients from the Czech Republic with higher *SELENOF* gene expression compared to low expression in tumor tissue. Subjects with higher *SELENOF* expression than median show poorer prognosis with shorter DFS and OS compared to subjects with lower *SELENOF* expression (50.9 vs. 65.2 months and 62.9 vs. 72.9 months, respectively; *PDFS* = 0.01 and *POS* = 0.021). In multivariable Cox regression analyses adjusted by tumor stage, surgical radicality, and chemotherapy this was associated with a Hazards ratio (HR) point estimate of 2.41 for DFS (95% CI: 1.08, 5.41; *P* = 0.032) and 4.13 (95% CI: 1.10, 15.39; *P* = 0.035) for OS.

Most of the studied genes (12 of the 17) showed non-significant expression changes in The Cancer Genome Atlas (TCGA) dataset (https://cancergenome.nih.gov/) (see Supplementary Figure S1). The other five genes showed concordant significant expression changes across the Irish, Czech, and the TCGA datasets, including the main Se transport protein-coding gene *SELENOP*, *GPX2*, *GPX3* (the second major Se transport gene), *TNRDX3,* and *SELENBP1* (see last column in Table 2). As presently we only had tissue RNA available for analysis, we were not able to analyze the protein expression in our samples. However, Supplementary Table S2 presents a summary of the immunohistochemistry protein expression data from the Human Protein Atlas (HPA) dataset (https://www.proteinatlas.org/), for the corresponding seventeen genes examined in this study. Although the numbers of the tumor samples with the available data are too small (*n* = 9–12 for each selenoprotein) to make meaningful inferences, several proteins (GPX2, GPX3, TXNRD3) had expression changes in most samples that matched the direction of gene expression variance observed in both the Irish and the Czech CRC samples. *SELENOP*, *SELENBP1*, and *SOD2* had medium protein expression levels, in both the normal

and the tumor cells. Contrary results were given only by *SELENOS* and *SEPHS2*, which appeared to have relatively higher protein expression levels in the tumor cells (although *SEPHS2* was not detected in the normal cells).

#### *3.3. Correlation of the Selenoprotein Transcript Levels with Se Status*

Spearman correlation coefficients were calculated to ascertain the correlation of the selenoprotein gene expression in the Irish cohort with two measures of serum Se status (Se and SELENOP); available for thirteen CRA and seventeen CRC (Se) and thirty-eight CRA and eighteen CRC (SELENOP) patients, respectively. Only positive correlations of gene expression with the Se level were observed for the *SELENOF*, *SELENOK*, and *TXNRD1* genes, all in the tumor tissue (*p* = 0.001, 0.004, and 0.04, respectively), except for the normal tissue expression of *SELENOK* as well (*p* = 0.03). Regarding the SELENOP level, *TXNRD2* and *TXNRD3* tumor tissue expression was negatively correlated (*p* = 0.037 and 0.045), while *GPX1* had a higher expression in the normal tissue, *p* = 0.034. The only significant findings for adenomas were negative correlations for the SELENOP levels with *TXNRD1* disease (*p* = 0.006) and *SELENOW* normal (*p* = 0.042) tissue expressions. All correlation values are available in Supplementary Table S3.

#### **4. Discussion**

Expression profiles of seventeen Se pathway genes (including fifteen selenoproteins) were assessed in adenoma and cancer tissues, with their respective matched normal tissues, for forty CRA and a hundred and twenty-seven CRC patients. In comparisons between the neoplastic and normal tissue pairs, we observed seven differentially expressed genes in the CRA patients from Ireland and twelve dysregulated Se pathway genes for the CRC patients from the Czech Republic. Genes up-regulated in the tumor tissues were *GPX1*, *GPX2*, *SELENOH*, *TXNRD3*, and *SOD2*, while those down-regulated included *GPX3*, *SELENOP*, *SELENOS*, *SEPHS2*, *SELENBP1*, *SELENON*, and *SELENOK*. In adenomas *GPX2*, *SELENOH*, and *TXNRD3* also exhibited a higher expression in the disease tissue, while *GPX3*, *SELENOP*, *SELENOS*, and *SEPHS2* showed a lower expression for the Czech cancers. Thus, *GPX1*, *SELENBP1*, *SELENON*, *SELENOK*, and *SOD2* were differently expressed only in the cancer tissues.

Broadly similar expression patterns were observed for several selenoprotein genes (e.g., *GPX2*, *GPX3*, *SELENOP*, *SELENBP1*, and *TNRDX3*) across our Irish and Czech study groups, as compared with the gene and protein data for the TCGA and HPA, respectively. There were non-significant expression changes in the other selenoprotein genes in the TCGA, compared to significant findings in all our sample sets (*SEPHS2*, *SELENOS*), although the trend for a lower expression in tumor tissue for these genes was also seen in the TCGA. However, this is discordant with the corresponding SEPHS2 and SELENOS protein data in the HPA. These findings are probably largely explained by differences in the baseline Se status of the heterogenous populations in the TCGA and HPA data, resulting in differential regulation within the Se hierarchy [8], the method used for gene expression analysis (the more insensitive RefSeq, compared to the qPCR, for subtle changes), and variances in expression regulation at the gene or protein level.

Selenoproteins have several well-demonstrated or suggested cell protection roles in pathways implicated in colorectal carcinogenesis, such as the antioxidant response, immune, and inflammatory pathways [25–27], while downstream-targeted metabolic pathways are also affected by Se status, as demonstrated in human rectal biopsies [9]. Thus, altered selenoprotein expression in the colorectal tract may also be affected by Se status could increase cancer development risk by weakening the gut epithelial cell response to harmful oxidative and inflammatory challenges [8]. Transcriptomics animal studies highlight *Gpx1*, *Selenof*, *Selenoh*, and *Selenow* as being sensitive to Se supply and human Single Nucleotide Polymorphism (SNP) studies suggest *SELENOP*, *SELENOS*, *GPX4*, *SELENOF*, *SELENON*, *SELENOH*, and *TXNRD1-3* are key selenoproteins for colonic function and colorectal carcinogenesis [8,16,27,28]. However, prior to our investigation, data were lacking on the expression of all these selenoproteins in CRA and CRC. In this study, changes in gene expression were observed

for *SELENOP*, *SELENOS*, and *TXNRD3* for all sample groups, while *GPX4* was significant in the Irish cancers only and *GPX1*, *SELENOH*, and *SELENON* were differently regulated in the Czech CRCs. Additional genes in this study, showing changes in gene expression for both the Irish and the Czech cancers were *GPX2*, *GPX3*, *SELENOK*, *SEPHS2*, *SELENBP1*, and *SOD2*.

Only a higher expression of the *SELENOF* gene was associated with patient outcomes from cancer (lower DFS and OS), although this was non-significant after multiple testing adjustment. However, several other lines of evidence suggest that *SELENOF* may act as an Se-dependent oncogenic protein. *SELENOF* can be regulated by both Se status (as indicated also in our study) and endoplasmic reticulum (ER) stress, and several *SELENOF* genetic variants have been associated with an altered risk at the different cancer sites, including CRC [8,29]. Notably, previous cell and mouse-model studies suggest that higher levels of *SELENOF* may potentially contribute to favorable growth conditions for the cancerous cells in the colorectum, by reducing cellular ER stress and nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) activation [29–31].

Normally, selenoprotein expression is sensitive to limited Se supply in a tissue-dependent manner [11]. As expected in conditions of redox stress, a lowered *SELENOP* expression was reported in studies of a small number of German CRA and CRC subjects, while heterogenous *GPX2* mRNA and protein expression was observed between the tumor samples [13,32]. In German patients, *GPX1*, *GPX4*, and *TXNRD1* were also found to be up-regulated in cancer, compared to the matched tissues for mRNA and/or their corresponding protein levels [14]. However, while *GPX1* and *GPX4* were more highly expressed in tumor tissue from the Czech and Irish cancer groups, respectively, *TXNRD1* was not significantly different in any of our tested cohorts. In other settings of generally higher Se availability, such as in Japan, the protein expression of GPX1, GPX3, and SELENOP was reported to be lower in the CRC tumors, whereas the less Se-sensitive GPX2 was increased [15]. We observed similar results for our mRNA assays for *GPX2*, *GPX3*, and *SELENOP* for all CRA and CRC groups.

Hypoxic and oxidative stresses in proliferating tumors may also decouple the normal hierarchy of selenoprotein expression [12]. *SELENOH* is a putative redox regulating DNA-binding protein, whose cellular expression is sensitive to Se supply [26,33]. In contrast, protein expression of SELENOH has recently been shown to be higher in CRC human tumors (and to control cell-cycle progression and tumor proliferation in mouse and human CRC cell-line models) [12], aligning with the upregulation of *SELENOH* in the CRAs and the Czech CRCs observed in this study. The Se binding protein 1 (*SELENBP1*) gene and corresponding protein expression was found to be downregulated in Chinese CRC samples, as observed for the *SELENBP1* mRNA levels in the cancer cohorts in this study [34].

Overall, the apparent expression pattern we assessed in the colorectal tumor tissue is that the genes related to Se homeostasis (*SELENOP*, *SEPHS2*, *SELENBP1*) and ER stress (*SELENOK*, *SELENOS*) are down-regulated, while the antioxidant enzymes might exhibit a higher (*GPX1*, *GPX2*, *SELENOH*, *TXNRD3*, *SOD2*) or lower (*GPX3*, *SELENON*) expression [3,6,8]. In a previous examination of the common genetic variation in selenoproteins and Se-pathway genes, we observed that several SNPs in many of these genes were associated with CRC risk [35]. These data all support a role of selenoprotein metabolism and endoplasmic and oxidative stress in CRC development. Differences and overlaps between studies likely reflect design issues of sample size and tissue stage and site sampled, or underlying biological differences in the Se metabolism. This could be due to population specific differential regulation within the Se hierarchy, depending on the Se availability and selenoprotein genetic variation [8].

Several selenoprotein genes showed sequential expression differences through the major stages of dysplastic adenoma progression observed in different individuals in the Irish study. These included increasing down-regulation of *GPX3*, *SELENOP*, *SELENOS*, and *SEPHS2* and up-regulation of *GPX2*, *SELENOH*, and *TXNRD3*, in groups of normal-matched tissues, adenomas, and adenomas with HGD. Additional and larger studies will help clarify the pattern of selenoprotein expression in the adenoma progression. Possibly, selenoprotein expression may have uses as markers of advanced adenoma

stages, relevant for CRC screening, especially as only advanced adenomas appear to be associated with an increased risk for subsequent CRC development [36].

Suboptimal Se status levels are found in many parts of Europe [37,38]. Our measures of Se status were only possible for available matching serum samples for the Irish CRA and CRC patients. Here, the mean levels of Se and *SELENOP* (86.1 μg/L in 30 sera and 5.1 mg/L in 56 sera, respectively) were slightly higher, as compared to eight other Western European countries in a prospective study, showing an association of higher levels with a decreased CRC risk (85.6 μg/L and 4.4 mg/L, respectively for the controls [7]). Although, there were no marked correlation patterns between either of these serum Se status levels and the selenoprotein gene expression, they did indicate the potential importance of the Se supply for expression of *GPX1*, *SELENOF*, *SELENOK*, and *TXNRD1-3*. Most of the few significant correlations were observed for the tumor tissue gene expression, rather than adenoma tissue or the matched normal tissues for these pathologies, suggesting that Se status is a factor for the differential expression we observed for *SELENOK* and *TXNRD3* in the Irish CRC group. Although we did not observe significant correlations of their expression with the Se status, other selenoprotein genes whose activities are thought to be sensitive to lower Se status in the gastrointestinal tract, such as *SELENOP* and *SELENOH* [8,12], were also observed to be differentially regulated in the adenomas and/or cancers groups from Ireland. However, the total numbers available for these analyses were small and confined to the Irish samples. Therefore, this should be considered as an exploratory analysis requiring further investigation to more fully ascertain the degree to which any involvement of selenoprotein expression in affecting CRA to CRC development may depend on the Se status of the studied population.

In summary, several selenoprotein genes were differentially expressed in both the CRA and CRC disease-normal tissue pairs. These include key biological stress response genes like *GPXs*, *TXNRD3*, *SELENOS*, *SELENOH*, and the related *SOD2* gene implicated in cancer cell survival, and genes such as *SELENOP* and *SEPHS2* involved in Se biosynthesis. However, as only some of these expression changes were correlated with the Se status levels, selenoprotein expression may affect CRA to CRC development independent of the Se status. Functional studies will be required to further investigate any role of the identified differentially-expressed selenoproteins in colorectal carcinogenesis. Potentially, selenoprotein mRNA expression may have uses as biomarkers of colorectal function, colorectal neoplasia progression, and improved assessment of physiological Se status, with implications for the modulation of Se intake.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2072-6643/10/11/1812/ s1, Figure S1: Selenoprotein gene expression profiles from tumor-normal data of human colorectal tumor (red boxes) and mucosa (grey boxes) tissues from the TCGA database, Table S1: List of TaqMan selenoprotein and selenium-related gene expression assays used for this study, Table S2: Protein levels of selenoproteins in normal and colorectal cancer tissues in The Human Protein Atlas, Table S3: Spearman correlation values for tissue selenoprotein gene expression and serum selenium status in cancer and adenoma patients from Ireland.

**Author Contributions:** Conceptualization, D.J.H. and P.S.; Data curation, T.K., L.S., V.L. and N.S.; Formal analysis, D.J.H., T.K., L.S. and P.S.; Funding acquisition, D.J.H. and P.S.; Investigation, D.J.H., T.K., L.S. and P.S.; Methodology, D.J.H., T.K., L.S., V.L. and P.S.; Project administration, D.J.H., V.L., N.S. and P.S.; Resources, V.L., N.S. and P.S.; Software, T.K., L.S. and P.S.; Supervision, D.J.H. and P.S.; Validation, D.J.H. and L.S.; Writing–original draft, D.J.H. and T.K.; Writing–review & editing, L.S., N.S. and P.S.

**Funding:** This research was funded by project grants HRA/PHS/2013/397 and HRA/PHS/2015/1142 (Health Research Board of Ireland, P.I., D.J.H.), P303/12/G163 (the Czech Science Foundation to P.S.), LO1503 (National Sustainability Program I of the Ministry of Education Youth and Sports of the Czech Republic to T.K.), UNCE/MED/006 (Charles University project "Center of clinical and experimental liver surgery" to V.L.), and FNPl 00669806 (Conceptual Development of Research Organization-Faculty Hospital in Pilsen to V.L. and P.S.). L.S. was supported by Deutsche Forschungsgemeinschaft (DFG Research Unit 2558 TraceAge, Scho 849/6-1).

**Conflicts of Interest:** The authors declare no conflict of interest. While L.S. is the founder of selenOmed GmbH, a company involved in improving Se diagnostics, the data were analyzed before the founding of selenOmed and the analyses were conducted completely blinded to any clinical finding. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

#### **References**

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).


**PERIPHERAL NEUROPATHY – ADVANCES IN DIAGNOSTIC AND THERAPEUTIC APPROACHES**

Edited by **Ghazala Hayat**

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http://dx.doi.org/10.5772/1276 Edited by Ghazala Hayat

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Jorge Guajardo-Rosas, Faride Chejne Gomez, Ricardo Plancarte Sanchez, Figen Guney, Kathrine Jáuregui-Renaud, Alexander Cardenas-Mejia, Xitlali Baron, Javier Lopez-Mendoza, Colin Coulter, Claudia Gutiérrez, Leigh Maria Ramos-Platt, Young-Sup Yoon, Julie Kim, Yuko Kanbayashi, Toyoshi Hosokawa, Yousuke Katsuda, Rick Dobrowsky, Chengyuan Li, Deirdre O'Connor, Thais Federici, Nicholas Boulis, Shunsuke Kobayashi, Kohji Takei

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